LACTOFERRICIN DERIVED PEPTIDES

Abstract

 Summary:
The present invention relates to an isolated peptide to be used in the treatment of cancer consisting of 12 to 50 amino acid residues comprising
- at least two beta-strands, or
- at least two alpha-helices or
- at least one beta-strand and at least one alpha-helix,

wherein said beta-strands and/or alpha-helices are separated from each other by at least one turn, wherein the peptide has a positive net charge of +7 or more.

Description

[0001] The present invention relates to an isolated peptide exhibiting antitumoral effects.

[0002] Every year millions of people are diagnosed with cancer worldwide. Notwithstanding in the last decades much progress has been achieved in cancer therapy, nevertheless cancer remains a leading cause of death. Nowadays, surgery, chemotherapy, radiation, hormone ablation therapy and targeted therapy are the standard treatments, but in the year 2008 these were not curative in more than 50% of the cases. Furthermore, the use of these types of therapy is limited due to resistance and is accompanied by potential toxicity and diverse side effects due to inadequate specificity for tumor cells. Obviously, the discovery of new and more specific targets, together with the design of specific antitumor drugs, is one of the major interests in cancer research.

[0003] Cancer cells are often well characterized, but little is known about the plasma cell membrane, or to be more precise, the arising differences in the lipid composition in carcinogenesis. Eukaryotic plasma membranes usually comprise an overall neutral charge on the outer leaflet due to the zwitterionic phosphatidylcholine (PC) and sphingomyelin (SM). The negatively charged phospholipid phosphatidylserine (PS) together with the major part of phosphatidylethanolamine (PE) normally only assembles in the inner leaflet of eukaryotic plasma membranes. This asymmetric distribution of phospholipids is well documented and is maintained by an ATP-dependent aminophospholipid translocase. This asymmetry can get lost due to exposure of the negatively charged phosphatidylserine on the surface of cancerous and other pathological cells, apoptotic cells, as well as platelets and erythrocytes upon activation.

[0004] Based on the knowledge of PS exposure, new strategies for the design of anticancer drugs can be considered, especially cationic host defense derived peptides interacting with negatively charged phospholipids. Host defense peptides have emerged as potential alternative anticancer therapeutics offering many advantages over other therapies. Because of their mode of action and specificity - the cell membrane being the major target - resistance and cytotoxicity are less likely to occur and thus, they are also expected to cause fewer side effects. Furthermore, these peptides mostly damage cell membranes within minutes, which would hinder formation of resistance. Host defense peptides being part of the innate immune system of many diverse species (e.g. mammals, insects, amphibians) were initially discovered because of their antimicrobial activity. Currently, the antimicrobial peptide database lists more than 100 natural host defense peptides with antitumor activity.

[0005] One prominent member of anticancer peptides is bovine lactoferricin (bLFcin), which is generated from lactoferrin through pepsin cleavage. bLFcin possesses an acyclic twisted antiparallel β-sheet structure due to a disulfide bridge between two cysteine residues. This peptide is able to inhibit liver and lung metastasis in mice. In vivo studies with bLFcin on fibrosarcoma, melanoma and colon carcinoma tumors revealed massive necrosis of the tumor tissue after exposure to the peptide (Yoo et al. Jpn.J Cancer Res. 88(1997):184-190). Furthermore, it is known that bLFcin inhibits the tumor growth of neuroblastoma xenografts in nude rats. Clarification of the mechanism revealed that bLFcin induces apoptosis in human tumor cells through a pathway mediated by production of the intracellular ROS and activation of Ca²⁺/Mg²⁺ -dependent endonucleases.

[0006] It is an object of the present invention to provide compounds and preparations which can be used to treat cancer.

[0007] Therefore the present invention relates to an isolated peptide to be used in the treatment of cancer consisting of 12 to 50 amino acid residues comprising

• at least two beta-strands, or
• at least two alpha-helices or
• at least one beta-strand and at least one alpha-helix,

wherein said beta-strands and/or alpha-helices are separated from each other by at least one turn, wherein the peptide has a positive net charge of +7 or more.

[0008] It turned out that peptides having a positive net charge of +7 and comprising at least two beta-strands or at least two alpha-helices or at least one beta-strand and at least one alpha-helix separated by at least one turn exhibit cytotoxic effects on cancerous/tumor cells in mammals. This means that the peptides of the present invention are able to affect the viability of such cells leading to their destruction. The cytotoxic effects of the peptides of the present invention are highly specific for cancerous/tumor cells. This means that these peptides affect healthy cells to a much lower extent (preferably to at least 10%, more preferably to at least 20%, even more preferably to at least 50%, in particular to at least 90 to 100%) compared to cancerous/tumor cells. This high specificity of the peptides of the present invention allows to treat mammals, in particular humans, with a much higher efficacy reducing commonly known side-effects regularly described for anti-cancer compounds. The cytotoxic effect of such compounds is usually unspecific resulting in the destruction not only of cancerous/tumor cells but also of healthy cells.

[0009] According to the present invention the at least two beta-strands or at least two alpha-helices or the at least one beta-strand or the at least one alpha-helix are separated by at least one turn resulting in peptides having the following general basic structures:

a) beta-strand - turn - beta-strand

b) alpha-helix - turn - alpha-helix

c) beta-strand - turn - alpha-helix

d) alpha-helix - turn - beta-strand

[0010] According to the present invention the peptides disclosed herein may also comprise 3, 4 or even 5 beta-strands or alpha-helices. In such a case the beta-strands or alpha-helices of the peptide can be grouped (e.g. two beta strands are located adjacent to each other) and separated by one or more (e.g. 2, 3 or 4) turns or every single strand or helix is separated by one or more turns. The isolated peptide of the present invention may therefore comprise also more than one stretches having the above general basic structure.

[0011] The peptides of the present invention have a positive net charge of +7 or more. This means that the peptides of the present invention may have preferably a positive net charge of +8, +9, +10, +11, +12, +13, +14, +15 or even of +20. A positive net charge of at least +7 of the peptides of the present invention results in a better adsorption to the target membrane (negatively charged) and better stabilization of the secondary structure by hydrogen bridges.

[0012] Alpha helix (α-helix) is a common motif in the secondary structure of proteins, polypeptides and peptides. Alpha helices have a right-handed coiled or spiral conformation, in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues. The beta sheet (β-sheet) is the second form of regular secondary structure in proteins, polypeptides and peptides. Beta-sheets consist of beta strands connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet. A turn is a structural motif where the Cα atoms of two residues separated by one or more peptide bonds are in close approach (approx. < 7 Å), while the corresponding residues do not form a regular secondary structure element such as an alpha-helix or beta-sheet. The secondary structure of putative membrane active dimer peptides can accurately be predicted by the online program PEP-FOLD: e.g. http://bioserv.rpbs.univ-paris-diderot.fr/PEP-FOLD/ (see Maupetit, J et al Nucleic Acids Res. 37(2009), W498-W503 and/or Thévenet P et al Nucleic Acid Res. 40(2012), W288-W293) or as described in Chou PY et al (Annual Review of Biochemistry 47(1978):251-276). Most preferably the method as described in Thévenet P et al is used to determine the secondary structure of the peptide of the present invention.

[0013] The person skilled in the art is able to identify peptides exhibiting the properties as described herein using known methods. The secondary structure can be identified as described above. The net charge can be calculated by summing up the positive and negative charges of the amino acid residues present in a peptide.

[0014] One skilled in the art can easily synthesize the peptides of the present invention. Standard procedures for preparing synthetic peptides are well known in the art. Peptides of the present invention can be synthesized by commonly used methods as t-BOC or FMOC protection, preferably FMOC protection, of alpha-amino groups. Both methods involve stepwise syntheses whereby a single amino acid is added at each step starting from the carboxyl-terminus of the peptide (See, Coligan et al., Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9). Peptides of the invention can also be synthesized by the solid phase peptide synthesis methods well known in the art. (Merrifield, J. Am. Chem. Soc., 85:2149, 1963), and Stewart and Young, Solid Phase Peptides Synthesis, Pierce, Rockford, Ill. (1984)). Peptides can be synthesized using a copoly(styrenedivinylbenzene) containing 0.1-1.0 mMol amines/g polymer. On completion of chemical synthesis, the peptides can be deprotected and cleaved from the polymer by treatment with liquid HF-10% anisole for about 0.25 to 1 hour at 0°C. After evaporation of the reagents, the peptides are extracted from the polymer with 1% acetic acid solution which is then lyophilized to yield the crude material. This can typically be purified by such techniques as gel filtration on Sephadex G-15 using 5% acetic acid as a solvent, by high pressure liquid chromatography, and the like. Lyophilization of appropriate fractions of the column will yield the homogeneous peptide or peptide derivatives, which can then be characterized by such standard techniques as amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopy, molar rotation, solubility, and assessed by the solid phase Edman degradation (see e.g Protein Purification, M. P. Deutscher, ed. Methods in Enzymology, Vol 182, Academic Press, 1990). Automated synthesis using FMOC solid phase synthetic methods can be achieved using an automated peptide synthesizer (Model 432A, Applied Biosystems, Inc.).

[0015] The peptides/polypeptides of the present invention can also be synthesized using a fusion protein microbial method in which an anionic carrier peptide is fused to a cationic peptide. A method for such microbial production of cationic peptides having anti-microbial activity is provided in US 5,593,866.

[0016] The peptides of the present invention thus produced can be purified by isolation/purification methods for proteins generally known in the field of protein chemistry. More particularly, there can be mentioned, for example, extraction, recrystallization, salting out with ammonium sulfate, sodium sulfate, etc., centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography, reversed-phase chromatography, gel filtration method, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution, etc. and combinations of these. Most effective is a method by reversed-phase high performance liquid chromatography.

[0017] The peptides of the present invention may form a salt by addition of an acid. Examples of the acid include inorganic acids (such as trifluoroacetic acid, hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, and sulfuric acid) or organic carboxylic acids (such as acetic acid, propionic acid, maleic acid, succinic acid, malic acid, citric acid, tartaric acid, and salicylic acid), acidic sugars such as glucuronic acid, galacturonic acid, gluconic acid, ascorbic acid, etc., acidic polysaccharides such as hyaluronic acid, chondroitin sulfates, alginic acid, or organic sulfonic acids (such as methanesulfonic acid, and p-toluenesulfonic acid), and the like. Of these salts, preferred is a pharmaceutically acceptable salt.

[0018] The peptides of the present invention may form a salt with a basic substance. Examples of the salt include, for example, pharmaceutically acceptable salts selected from salts with inorganic bases such as alkali metal salts (sodium salt, lithium salt, potassium salt etc.), alkaline earth metal salts, ammonium salts, and the like or salts with organic bases, such as diethanolamine salts, cyclohexylamine salts and the like.

[0019] The term "amino acid" and "amino acid residue" as used herein means L-amino acids. However, also D-amino acids may be employed in the manufacturing of the peptides according to the present invention.

[0020] The peptide of the present invention preferably exhibits amphipathic properties. This means that the peptide of the present invention may comprise hydrophobic and hydrophilic regions. Methods to determine amphipathic properties are well known in the art.

[0021] According to a preferred embodiment of the present invention the isolated peptide comprises at least one peptide having amino acid sequence (X₁)_M-X₂-(X₃)_P-X₄-(X₅)_Q-X₆-(X₇)_S (SEQ ID No. 203) or the reverse sequence thereof, wherein
X₁ is a hydrophobic amino acid, preferably selected from the group consisting of phenylalanine (Phe), alanine (Ala), leucine (Leu) and valine (Val),
X₂ is a hydrophobic amino acid, preferably tryptophan (Trp),
X₃ is selected from the group consisting of alanine (Ala), arginine (Arg), glutamine (Gln), asparagine (Asn), proline (Pro), isoleucine (Ile), leucine (Leu) and valine (Val),
X₄ is selected from the group consisting of isoleucine (Ile), phenylalanine (Phe), tryptophan (Trp) and tyrosine (Tyr),
X₅ is selected from the group consisting of arginine (Arg), lysine (Lys), tyrosine (Tyr) and phenylalanine (Phe),
X₆ is a hydrophobic amino acid, preferably selected from the group consisting of isoleucine (Ile), tryptophan (Trp), valine (Val) and leucine (Leu), and
X₇ is selected from the group consisting of arginine (Arg), lysine (Lys), isoleucine (Ile) and serine (Ser), and wherein
M is 1 or 2,
Q is 1 or 2,
P is 2 or 3, and
S is 1, 2, 3 or 4 under the proviso that if (X₅)_Q is Arg-Arg S is 1.

[0022] As used herein the term "reverse sequence" of an amino acid sequence means that a specific sequence is reversed. For instance, the reverse sequence of the amino acid sequence ABCDEFG is GFEDCBA.

[0023] The at least one peptide has an amino acid sequence selected from the group consisting of FWQRIRKVR (SEQ ID No. 1), FWQRRIRKVRR (SEQ ID No. 2), FWQRKIRKVRK (SEQ ID No. 3), FWQRNIRIRR (SEQ ID No. 4), FWQRNIRKVR (SEQ ID No. 5), FWQRNIRVR (SEQ ID No. 6), FWQRNIRKVRR (SEQ ID No. 7), FWQRNIRKVKK (SEQ ID No. 8), FWQRNIRKVRRR (SEQ ID No. 9), FWQRNIRKVKKK (SEQ ID No. 10), FWQRNIRKVRRRR (SEQ ID No. 11), FWQRNIRKVRRRI (SEQ ID No. 12), FWQRNIRKVKKKK (SEQ ID No. 13), FWQRNIRKVKKKI (SEQ ID No. 14), FWQRNIRKIR (SEQ ID No. 15), FWQRNIRKLR (SEQ ID No. 16), FWQRNIRKWR (SEQ ID No. 17), FWQRNWRKVR (SEQ ID No. 18), FWQRNFRKVR (SEQ ID No. 19), FWQRNYRKVR (SEQ ID No. 20), FWQRNIRKVS (SEQ ID No. 21), FWQRRIRIRR (SEQ ID No. 22), FWQRPIRKVR (SEQ ID No. 23), FWQRRIRKWR (SEQ ID No. 24), FWPRNIRKVR (SEQ ID No. 26), FWARNIRKVR (SEQ ID No. 27), FWIRNIRKVR (SEQ ID No. 28), FWLRNIRKVR (SEQ ID No. 29), FWVRNIRKVR (SEQ ID No. 30), FWQRNIFKVR (SEQ ID No. 31), FWQRNIYKVR (SEQ ID No. 32), FAWQRNIRKVR (SEQ ID No. 33), FLWQRNIRKVR (SEQ ID No. 35) and FVWQRNIRKVR (SEQ ID No. 36) or the reverse sequence thereof.

[0024] According to another preferred embodiment of the present invention the isolated peptide comprises at least one peptide having amino acid sequence (X_1')_M' - X_2' - (X_3')_P' - (X_4')_Q' - (X_5')_T' - (X_6')_R' -(X_7')_S' (SEQ ID No. 204) or the reverse sequence thereof, wherein
X_1' is a hydrophobic amino acid, preferably selected from the group consisting of phenylalanine (Phe) and isoleucine (Ile),
X_2' is a hydrophobic amino acid, preferably tryptophan (Trp),
X_3' is selected from the group consisting of glycine (Gly), asparagine (Asn), isoleucine (Ile) and phenylalanin (Phe),
X_4' is isoleucine (Ile) or tryptophan (Trp),
X_5' is arginine (Arg) or lysine (Lys),
X_6' is a hydrophobic amino acid, preferably selected from the group consisting of isoleucine (Ile), tryptophan (Trp) and valine (Val) and
X_7' is arginine (Arg), and wherein
M' is 1 or 2,
T' is 1 or 2,
R' is 0 or 1,
P' is 1, 2 or 3,
Q' is 1, and
S' is 0, 1 or 2.

[0025] The at least one peptide of the present invention may have an amino acid sequence selected from the group consisting of FWRIRKWR (SEQ ID No. 37), FWRIRKVR (SEQ ID No. 38), FWRWRR (SEQ ID No. 39), FWRRWRR (SEQ ID No. 40), FWRRWIRR (SEQ ID No. 41), FWRGWRIRR (SEQ ID No. 42), FWRRFWRR (SEQ ID No. 43), FWRWRWR (SEQ ID No. 44), FWRIWRWR (SEQ ID No. 45), FWRIWRIWR (SEQ ID No. 46), FWRNIRKWR (SEQ ID No. 47) and FWRRRIRIRR (SEQ ID No. 48) or the reverse sequence thereof.

[0026] According to a further preferred embodiment of the present invention the isolated peptide comprises at least one peptide having amino acid sequence (X_1")_M" - X_2" - (X_3")_P" - (X_4")_Q" - (X_5")_R" -(X_6")_S" (SEQ ID No. 205) or the reverse sequence thereof, wherein
X_1" is a hydrophobic amino acid, preferably selected from the group consisting of proline (Pro) and phenylalanine (Phe),
X_2" is a hydrophobic amino acid, preferably tryptophan (Trp),
X_3" is selected from the group consisting of alanine (Ala), arginine (Arg), glutamine (Gln), lysine (Lys), tryptophan (Trp) and isoleucine (Ile),
X_4" is selected from the group consisting of arginine (Arg) and aspartate (Asp),
X_5" is a hydrophobic amino acid, preferably selected from the group consisting of isoleucine (Ile), tryptophan (Trp), phenylalanine (Phe), valine (Val) and leucine (Leu), and
X_6" is selected from the group consisting of arginine (Arg), lysine (Lys), isoleucine (Ile), serine (Ser) and aspartate (Asp), and wherein
M" is 0, 1, 2 or 3,
Q" is 0, 1, 2 or 3,
R" is 1 or 2,
P" is 1, 2 or 3, and
S" is 1, 2 or 3.

[0027] The at least one peptide has preferably an amino acid sequence selected from the group consisting of PFWRWRIWR (SEQ ID No. 50), PFWRIRIRR (SEQ ID No. 51), PFWRQRIRR (SEQ ID No. 52), PFWRARIRR (SEQ ID No. 53), PFWRKRIRR (SEQ ID No. 54), PFWRKRLRR (SEQ ID No. 55), PFWRKRWRR (SEQ ID No. 56), PFWRRRIRR (SEQ ID No. 57), PFWRRRWRR (SEQ ID No. 58), PFWRIRIRRD (SEQ ID No. 59), PFFWRIRIRR (SEQ ID No. 60), PWRIRIRR (SEQ ID No. 61), PFWRRQIRR (SEQ ID No. 81), PFWRKKLKR (SEQ ID No. 82), PWRRIRR (SEQ ID No. 83), PWRRKIRR (SEQ ID No. 84) and PFWRRIRIRR (SEQ ID No. 85) or the reverse sequence thereof.

[0028] According to a preferred embodiment of the present invention the isolated peptide comprises at least one peptide having amino acid sequence (X_1"')_M"'-(X_2"')_O"'-X_3"' - (X_4"')_P"' - (X_5"')_Q"' - (X_6"')_T"' - (X_7"')_R"' - (X_8"')_S"' (SEQ ID No. 206) or the reverse sequence thereof, wherein
X_1"' is a hydrophobic amino acid, preferably selected from the group consisting of proline (Pro) and phenylalanine (Phe),
X_2"' is a basic amino acid, preferably arginine (Arg),
X_3"' is a hydrophobic amino acid, preferably tryptophan (Trp),
X_4"' is selected from the group consisting of alanine (Ala), arginine (Arg), glutamine (Gln), asparagine (Asn) and lysine (Lys),
X_5"' is selected from the group consisting of isoleucine (Ile), phenylalanine (Phe) and tryptophan (Trp),
X_6"' is selected from the group consisting of glutamine (Gln), arginine (Arg) and asparagine (Asn),
X_7"' is a hydrophobic amino acid, preferably selected from the group consisting of isoleucine (Ile), tryptophan (Trp) and phenylalanine (Phe), and
X_8"' is arginine (Arg), and wherein
M"' is 0, 1, 2 or 3,
T"' is 0, 1, 2 or 3,
O"' is 0 or 1,
P"' is 1, 2 or 3,
Q"' is 1 or 2, and
R"' and S"' are 0, 1 or 2.

[0029] The at least one peptide may have an amino acid sequence selected from the group consisting of FWRNIRIRR (SEQ ID No. 72), FWQRIRIRR (SEQ ID No. 73), FWRWRIWR (SEQ ID No. 74), FWRIRIRR (SEQ ID No. 75), FWRNIRIWRR (SEQ ID No. 76) and FWRNIRIRR (SEQ ID No. 77) or the reverse sequence thereof.

[0030] The isolated peptide comprises preferably at least one peptide having an amino acid sequence selected from the group consisting of RFWQRNIRKVRR (SEQ ID No. 62), RFWQRNIRKYR (SEQ ID No. 63), PFWQRNIRKWR (SEQ ID No. 64), RFRWQRNIRKYRR (SEQ ID No. 65), RWKRINRQWF (SEQ ID No. 66), KRFCFKK (SEQ ID No. 67), KRFSFKKC (SEQ ID No. 68), KRWSWKK (SEQ ID No. 69), FRFSFKK (SEQ ID No. 70), RRFWFRR (SEQ ID No. 71), RFWQRNIRIRR (SEQ ID No. 78), RWQRNIRIRR (SEQ ID No. 79) and RRWFWRR (SEQ ID No. 86) or the reverse sequence thereof.

[0031] According to a further embodiment of the present invention the isolated peptide comprises at least one peptide having an amino acid sequence selected from the group consisting of FIWQRNIRKVR (SEQ ID No. 34), FIWRWRWR (SEQ ID No. 49) and RRIRINRQWF (SEQ ID No. 80) or the reverse sequence thereof.

[0032] The isolated peptide of the present invention may comprise a single peptide or the reverse sequence thereof as defined above. However, the isolated peptide may also comprise a multiplicity (at least two, at least three, at least four etc.) of said single peptides or peptides having the reversed sequence thereof. According to a very particular preferred embodiment of the present invention the isolated peptide comprises at least two, most preferably two, of the aforementioned peptides.

[0033] According to a particularly preferred embodiment of the present invention the peptide of the present invention is selected from the peptides as shown in Table 1 having at least two beta-strands, or at least two alpha-helices or at least one beta-strand and at least one alpha-helix, said beta-strands and/or alpha-helices being separated from each other by at least one turn. Preferred are those peptides having an amino acid sequence selected from the group consisting of SEQ ID. No. 89, SEQ ID. No. 91, SEQ ID. No. 93, SEQ ID. No. 95, SEQ ID. No. 97, SEQ ID. No. 98, SEQ ID. No. 99, SEQ ID. No. 101, SEQ ID. No. 103, SEQ ID. No. 105, SEQ ID. No. 106, SEQ ID. No. 107, SEQ ID. No. 109, SEQ ID. No. 111, SEQ ID. No. 114, SEQ ID. No. 116, SEQ ID. No. 117, SEQ ID. No. 118, SEQ ID. No. 120, SEQ ID. No. 122, SEQ ID. No. 123, SEQ ID. No. 124, SEQ ID. No. 125, SEQ ID. No. 126, SEQ ID. No. 127, SEQ ID. No. 128, SEQ ID. No. 129, SEQ ID. No. 130, SEQ ID. No. 131, SEQ ID. No. 132, SEQ ID. No. 134, SEQ ID. No. 136, SEQ ID. No. 137, SEQ ID. No. 138, SEQ ID. No. 139, SEQ ID. No. 140, SEQ ID. No. 141, SEQ ID. No. 142, SEQ ID. No. 143, SEQ ID. No. 144, SEQ ID. No. 145, SEQ ID. No. 146, SEQ ID. No. 148, SEQ ID. No. 149, SEQ ID. No. 150, SEQ ID. No. 156, SEQ ID. No. 153, SEQ ID. No. 154, SEQ ID. No. 156, SEQ ID. No. 158, SEQ ID. No. 160, SEQ ID. No. 162, SEQ ID. No. 164, SEQ ID. No. 166, SEQ ID. No. 168, SEQ ID. No. 170, SEQ ID. No. 172, SEQ ID. No. 174, SEQ ID. No. 176, SEQ ID. No. 178, SEQ ID. No. 180, SEQ ID. No. 181, SEQ ID. No. 182, SEQ ID. No. 183, SEQ ID. No. 184, SEQ ID. No. 186, SEQ ID. No. 188, SEQ ID. No. 190, SEQ ID. No. 192, SEQ ID. No. 194, SEQ ID. No. 195, SEQ ID. No. 197, SEQ ID. No. 199, SEQ ID. No. 200, SEQ ID. No. 201 and SEQ ID. No. 202. The variable "X" within these sequences can be 1 to 3 (i.e. 1, 2 or 3) glycine or proline residues, preferably 1 proline residue. Particularly preferred peptides are those having a sequence selected from the group consisting of SEQ ID. No. 125, SEQ ID. No. 126, SEQ ID. No. 127, SEQ ID. No. 128, SEQ ID. No. 105, SEQ ID. No. 106, SEQ ID. No. 107, SEQ ID. No. 174, SEQ ID. No. 176, SEQ ID. No. 141, SEQ ID. No. 142, SEQ ID. No. 143, SEQ ID. No. 144, SEQ ID. No. 181, SEQ ID. No. 182, SEQ ID. No. 183 and SEQ ID. No. 184.

[0034] According to a preferred embodiment of the present invention the peptide of the present invention has/comprises/consists of a sequence selected from the group consisting of SEQ ID. No. 125, SEQ ID. No. 127, SEQ ID. No. 128, SEQ ID. No. 106 and SEQ ID. No. 141.

[0035] Of course the isolated peptide of the present invention may also comprise a combination of at least two of the aforementioned peptides.

[0036] According to a preferred embodiment of the present invention at least two peptides having an amino acid sequence as defined herein or the reverse sequence thereof are fused directly or via a linker, wherein said linker is preferably part of the turn, to each other.

[0037] According to a further preferred embodiment of the present invention said isolated peptide comprises at least two, preferably two, peptides having the same amino acid sequence and being selected from the peptides having an amino acid sequence as defined above or the reverse sequence thereof, wherein the at least two peptides are fused directly or via a linker to each other.

[0038] Said isolated peptide may comprise at least two, preferably two, peptides, wherein an at least one first peptide has an amino acid sequence as defined above and the at least one second peptide the reverse sequence thereof, wherein the at least two peptides are fused directly or via a linker to each other.

[0039] The linker comprises preferably 1 to 10, preferably 1 to 8, more preferably 1 to 5, even more preferably 1 to 3, amino acid residues.

[0040] According to a particularly preferred embodiment of the present invention the linker, being preferably part of the turn, comprises or consists of proline and/or glycine, preferably proline.

[0041] The isolated peptide of the present invention or a pharmaceutical preparation comprising at least one of said isolated peptides can be used to treat cancer of solid and non-solid tumors, including metastases, whereby the cancer is preferably selected from the group consisting of melanoma, rhabdomyosarcoma, glioblastoma, colorectal cancer, breast cancer, lymphoma, prostate cancer, pancreatic cancer, renal cancer, ovarian cancer and lung cancer. Particularly preferred cancers to be treated with the peptides of the present invention are glioblastoma and melanoma, preferably malignant melanoma. The peptides of the present invention is preferably administered to a patient in need thereof in an amount of 100µg/kg body weight to 100mg/kg body weight, preferably 1mg/kg body weight to 50mg/kg body weight, more preferably 5mg/kg body weight to 15mg/kg body weight, in particular 10mg/kg body weight. Furthermore the peptides of the present invention are preferably administered daily (e.g. three times a day, twice a day or once a day), every 2^nd, every 3^rd, every 4^th or every 5^th day.

[0042] In order to obtain a pharmaceutical composition with even better anti-cancer or anti-tumor activity additional agents exhibiting similar properties as the peptides according to the present invention are added. Of course it is also possible to add agents with activities other than the peptides according to the present invention. These substances may be helpful in increasing the bioavailability such as for example increasing the stability of the peptides or their delivery.

[0043] Such compositions according to the present invention may preferably further comprise a pharmaceutically acceptable excipient.

[0044] The pharmaceutical composition of the present invention may consist of the peptide of the present invention alone or may be in the form of a composition comprising the peptide of the present invention and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier which can be used is not limited particularly and includes an excipient, a binder, a lubricant, a colorant, a disintegrant, a buffer, an isotonic agent, a preservative, an anesthetic, and the like which can be used in a medical field.

[0045] The composition of the present invention can be administered, depending on the cancer to be treated locally or systemically by injection (subcutaneous, intracutaneous, intravenous, intraperitoneal, etc.), eye dropping, instillation, percutaneous administration, oral administration, inhalation, etc. The peptides of the present invention can also be directly injected into the tumor/cancer to be treated. This latter type of administration is particularly preferred when treating membrane.

[0046] Also, the dosage form such as injectable preparations (solutions, suspensions, emulsions, solids to be dissolved when used, etc.), tablets, capsules, granules, powders, liquids, liposome inclusions, ointments, gels, external powders, sprays, inhalating powders, eye drops, eye ointment, suppositories, pessaries, and the like can be appropriately selected depending on the administration method, and the composition of the present invention can be accordingly formulated.

[0047] Another aspect of the present invention relates to the use of a peptide as defined above for the manufacturing of a medicament for treating cancer in a mammal, in particular in a human patient.

[0048] A further aspect of the present invention relates to a method for treating a mammal, in particular a human patient, suffering from cancer by administering to said mammal an effective amount of a peptide as defined above.

[0049] As used herein, the term "therapeutically effective amount" or "effective amount" means that to a mammal an amount of the peptide of the present invention is administered which allows the reduction of the tumor cells within the body of at least 10%, preferably at least 20%, more preferably at least 50%, and more preferably sufficient to reduce by 90%. Generally, the dosage will vary with age, condition and sex, and can be determined by one skilled in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. In any event, the effectiveness of treatment can be determined by monitoring the presence of the cancer cells within the body.

[0050] According to a particularly preferred embodiment of the present invention the peptide of the present invention has/comprises/consists of a sequence selected from the group consisting of SEQ ID. No. 125, SEQ ID. No. 127, SEQ ID. No. 128, SEQ ID. No. 106 and SEQ ID. No. 141 and is used in the treatment of glioblastoma and melanoma, preferably malignant melanoma. The peptide of the present invention can be administered by directly injecting the peptide into the tumor/cancer.

[0051] Preferably the present invention is defined as in the following embodiments:

1. An isolated peptide to be used in the treatment of cancer consisting of 12 to 50 amino acid residues comprising

• at least two beta-strands, or
• at least two alpha-helices or
• at least one beta-strand and at least one alpha-helix,

wherein said beta-strands and/or alpha-helices are separated from each other by at least one turn, wherein the peptidehas a positive net charge of +7 or more.

2. Peptide for use according to embodiment 1, wherein the peptide comprises at least 5 hydrophobic amino acid residues.

3. Peptide for use according to embodiment 1 or 2, wherein the isolated peptide comprises at least one peptide having amino acid sequence (X₁)_M-X₂-(X₃)_P-X₄-(X₅)_Q-X₆-(X₇)_S or the reverse sequence thereof, wherein
X₁ is a hydrophobic amino acid, preferably selected from the group consisting of phenylalanine (Phe), alanine (Ala), leucine (Leu) and valine (Val),
X₂ is a hydrophobic amino acid, preferably tryptophan (Trp),
X₃ is selected from the group consisting of alanine (Ala), arginine (Arg), glutamine (Gln), asparagine (Asn), proline (Pro), isoleucine (Ile), leucine (Leu) and valine (Val),
X₄ is selected from the group consisting of isoleucine (Ile), phenylalanine (Phe), tryptophan (Trp) and tyrosine (Tyr),
X₅ is selected from the group consisting of arginine (Arg), lysine (Lys), tyrosine (Tyr) and phenylalanine (Phe),
X₆ is a hydrophobic amino acid, preferably selected from the group consisting of isoleucine (Ile), tryptophan (Trp), valine (Val) and leucine (Leu), and
X₇ is selected from the group consisting of arginine (Arg), lysine (Lys), isoleucine (Ile) and serine (Ser), and wherein
M is 1 or 2,
Q is 1 or 2,
P is 2 or 3, and
S is 1, 2, 3 or 4 under the proviso that if (X₅)_Q is Arg-Arg S is 1.

4. Peptide for use according to embodiment 3, wherein the at least one peptide have an amino acid sequence selected from the group consisting of FWQRIRKVR (SEQ ID No. 1), FWQRRIRKVRR (SEQ ID No. 2), FWQRKIRKVRK (SEQ ID No. 3), FWQRNIRIRR (SEQ ID No. 4), FWQRNIRKVR (SEQ ID No. 5), FWQRNIRVR (SEQ ID No. 6), FWQRNIRKVRR (SEQ ID No. 7), FWQRNIRKVKK (SEQ ID No. 8), FWQRNIRKVRRR (SEQ ID No. 9), FWQRNIRKVKKK (SEQ ID No. 10), FWQRNIRKVRRRR (SEQ ID No. 11), FWQRNIRKVRRRI (SEQ ID No. 12), FWQRNIRKVKKKK (SEQ ID No. 13), FWQRNIRKVKKKI (SEQ ID No. 14), FWQRNIRKIR (SEQ ID No. 15), FWQRNIRKLR (SEQ ID No. 16), FWQRNIRKWR (SEQ ID No. 17), FWQRNWRKVR (SEQ ID No. 18), FWQRNFRKVR (SEQ ID No. 19), FWQRNYRKVR (SEQ ID No. 20), FWQRNIRKVS (SEQ ID No. 21), FWQRRIRIRR (SEQ ID No. 22), FWQRPIRKVR (SEQ ID No. 23), FWQRRIRKWR (SEQ ID No. 24), FWPRNIRKVR (SEQ ID No. 26), FWARNIRKVR (SEQ ID No. 27), FWIRNIRKVR (SEQ ID No. 28), FWLRNIRKVR (SEQ ID No. 29), FWVRNIRKVR (SEQ ID No. 30), FWQRNIFKVR (SEQ ID No. 31), FWQRNIYKVR (SEQ ID No. 32), FAWQRNIRKVR (SEQ ID No. 33), FLWQRNIRKVR (SEQ ID No. 35) and FVWQRNIRKVR (SEQ ID No. 36) or the reverse sequence thereof.

5. Peptide for use according to any one of embodiments 1 to 4, wherein the isolated peptide comprises at least one peptide having amino acid sequence (X_1')_M' - X_2' - (X_3')_P' - (X_4')_Q'-(X_5')_T' - (X_6')_R' -(X_7')_S' or the reverse sequence thereof, wherein
X_1' is a hydrophobic amino acid, preferably selected from the group consisting of phenylalanine (Phe) and isoleucine (Ile),
X_2' is a hydrophobic amino acid, preferably tryptophan (Trp),
X_3' is selected from the group consisting of glycine (Gly), asparagine (Asn), isoleucine (Ile) and phenylalanin (Phe),
X_4' is isoleucine (Ile) or tryptophan (Trp),
X_5' is arginine (Arg) or lysine (Lys),
X_6' is a hydrophobic amino acid, preferably selected from the group consisting of isoleucine (Ile), tryptophan (Trp) and valine (Val) and
X_7' is arginine (Arg), and wherein
M' is 1 or 2,
T' is 1 or 2,
R' is 0 or 1,
P' is 1, 2 or 3,
Q' is 1, and
S' is 0, 1 or 2.

6. Peptide for use according to embodiment 5, wherein the at least one peptide have an amino acid sequence selected from the group consisting of FWRIRKWR (SEQ ID No. 37), FWRIRKVR (SEQ ID No. 38), FWRWRR (SEQ ID No. 39), FWRRWRR (SEQ ID No. 40), FWRRWIRR (SEQ ID No. 41), FWRGWRIRR (SEQ ID No. 42), FWRRFWRR (SEQ ID No. 43), FWRWRWR (SEQ ID No. 44), FWRIWRWR (SEQ ID No. 45), FWRIWRIWR (SEQ ID No. 46), FWRNIRKWR (SEQ ID No. 47) and FWRRRIRIRR (SEQ ID No. 48) or the reverse sequence thereof.

7. Peptide for use according to any one of embodiments 1 to 6, wherein the isolated peptide comprises at least one peptide having amino acid sequence (X_1")_M" - X_2" - (X_3")_P" - (X_4")_Q" - (X_5")_R" - (X_6")_S" or the reverse sequence thereof, wherein
X_1" is a hydrophobic amino acid, preferably selected from the group consisting of proline (Pro) and phenylalanine (Phe),
X_2" is a hydrophobic amino acid, preferably tryptophan (Trp),
X_3" is selected from the group consisting of alanine (Ala), arginine (Arg), glutamine (Gln), lysine (Lys), tryptophan (Trp) and isoleucine (Ile),
X_4" is selected from the group consisting of arginine (Arg) and aspartate (Asp),
X_5" is a hydrophobic amino acid, preferably selected from the group consisting of isoleucine (Ile), tryptophan (Trp), phenylalanine (Phe), valine (Val) and leucine (Leu), and
X_6" is selected from the group consisting of arginine (Arg), lysine (Lys), isoleucine (Ile), serine (Ser) and aspartate (Asp), and wherein
M" is 0, 1, 2 or 3,
Q" is 0, 1, 2 or 3,
R" is 1 or 2,
P" is 1, 2 or 3, and
S" is 1, 2 or 3.

8. Peptide for use according to embodiment 7, wherein the at least one peptide have an amino acid sequence selected from the group consisting of PFWRWRIWR (SEQ ID No. 50), PFWRIRIRR (SEQ ID No. 51), PFWRQRIRR (SEQ ID No. 52), PFWRARIRR (SEQ ID No. 53), PFWRKRIRR (SEQ ID No. 54), PFWRKRLRR (SEQ ID No. 55), PFWRKRWRR (SEQ ID No. 56), PFWRRRIRR (SEQ ID No. 57), PFWRRRWRR (SEQ ID No. 58), PFWRIRIRRD (SEQ ID No. 59), PFFWRIRIRR (SEQ ID No. 60), PWRIRIRR (SEQ ID No. 61), PFWRRQIRR (SEQ ID No. 81), PFWRKKLKR (SEQ ID No. 82), PWRRIRR (SEQ ID No. 83), PWRRKIRR (SEQ ID No. 84) and PFWRRIRIRR (SEQ ID No. 85) or the reverse sequence thereof.

9. Peptide for use according to any one of embodiments 1 to 8, wherein the isolated peptide comprises at least one peptide having amino acid sequence (X_1"')_M"' - (X_2"')_O"' - X_3"' - (X_4"')_P"' - (X_5"')_Q"'-(X_6"')_T"'-(X_7"')_R"'-(X_8"')_S"' or the reverse sequence thereof, wherein
X_1"' is a hydrophobic amino acid, preferably selected from the group consisting of proline (Pro) and phenylalanine (Phe),
X_2"' is a basic amino acid, preferably arginine (Arg),
X_3"' is a hydrophobic amino acid, preferably tryptophan (Trp),
X_4"' is selected from the group consisting of alanine (Ala), arginine (Arg), glutamine (Gln), asparagine (Asn) and lysine (Lys),
X_5"' is selected from the group consisting of isoleucine (Ile), phenylalanine (Phe) and tryptophan (Trp),
X_6"' is selected from the group consisting of glutamine (Gln), arginine (Arg) and asparagine (Asn),
X_7"' is a hydrophobic amino acid, preferably selected from the group consisting of isoleucine (Ile), tryptophan (Trp) and phenylalanine (Phe), and
X_8"' is arginine (Arg), and wherein
M"' is 0, 1, 2 or 3,
T"' is 0, 1, 2 or 3,
O"' is 0 or 1,
P"' is 1, 2 or 3,
Q"' is 1 or 2, and
R"' and S"' are 0, 1 or 2.

10. Peptide for use according to embodiment 9, wherein the at least one peptide have an amino acid sequence selected from the group consisting of FWRNIRIRR (SEQ ID No. 72), FWQRIRIRR (SEQ ID No. 73), FWRWRIWR (SEQ ID No. 74), FWRIRIRR (SEQ ID No. 75), FWRNIRIWRR (SEQ ID No. 76) and FwRNIRIRR (SEQ ID No. 77) or the reverse sequence thereof.

11. Peptide for use according to any one of embodiments 1 to 10, wherein the isolated peptide comprises at least one peptide having an amino acid sequence selected from the group consisting of RFWQRNIRKVRR (SEQ ID No. 62), RFWQRNIRKYR (SEQ ID No. 63), PFWQRNIRKWR (SEQ ID No. 64), RFRWQRNIRKYRR (SEQ ID No. 65), RWKRINRQWF (SEQ ID No. 66), KRFCFKK (SEQ ID No. 67), KRFSFKKC (SEQ ID No. 68), KRWSWKK (SEQ ID No. 69), FRFSFKK (SEQ ID No. 70), RRFWFRR (SEQ ID No. 71), RFWQRNIRIRR (SEQ ID No. 78), RWQRNIRIRR (SEQ ID No. 79) and RRWFWRR (SEQ ID No. 86) or the reverse sequence thereof.

12. Peptide for use according to any one of embodiments 1 to 11, wherein the isolated peptide comprises at least one peptide having an amino acid sequence selected from the group consisting of FIWQRNIRKVR (SEQ ID No. 34), FIWRWRWR (SEQ ID No. 49) and RRIRINRQWF (SEQ ID No. 80) or the reverse sequence thereof.

13. Peptide for use according to any one of embodiments 1 to 12, wherein at least two peptides having an amino acid sequence as defined in any one of embodiments 4 to 13 or the reverse sequence thereof are fused directly or via a linker to each other.

14. Peptide for use according to any one of embodiments 1 to 12, wherein said isolated peptide comprises at least two, preferably two, peptides having the same amino acid sequence and being selected from the peptides having an amino acid sequence as defined in any one of embodiments 4 to 13 or the reverse sequence thereof, wherein the at least two peptides are fused directly or via a linker to each other.

15. Peptide for use according to any one of embodiments 1 to 12, wherein said isolated peptide comprises at least two, preferably two, peptides, wherein an at least one first peptide has an amino acid sequence as defined in any one of embodiments 4 to 13 and the at least one second peptide the reverse sequence thereof, wherein the at least two peptides are fused directly or via a linker to each other.

16. Peptide for use according to any one of embodiments 13 to 15, wherein the linker comprises 1 to 10, preferably 1 to 8, more preferably 1 to 5, even more preferably 1 to 3, amino acid residues.

17. Peptide for use according to any one of embodiments 13 to 16, wherein the linker comprises proline and/or glycine residues.

18. Peptide for use according to any one of embodiments 13 to 17, wherein the linker consists of one, two or three, preferably one, proline residue.

19. Peptide for use according to any one of embodiments 1 to 18, wherein the cancer is selected from solid and non-solid tumors, including metastases.

20. Peptide for use according to any one of embodiments 1 to 19, wherein the cancer is selected from the group consisting of melanoma, rhabdomyosarcoma, glioblastoma, colorectal cancer, breast cancer, lymphoma, prostate cancer, pancreatic cancer, renal cancer, ovarian cancer and lung cancer.

21. Use of a peptide as defined in any one of embodiments 1 to 18 for the manufacturing of a medicament for treating cancer in a mammal, in particular in a human patient.

22. Method for treating a mammal, in particular a human patient, suffering from cancer by administering to said mammal an effective amount of a peptide as defined in any one of embodiments 1 to 18.

[0052] The present invention is further illustrated by the following figures and examples, however, without being restricted thereto.

Fig. 1 shows the secondary structures of PEP-322 (A) and R-DIM-P-PEP-322 (B) in Hepes buffer (first bar) or presence of SDS and DPC at peptide to surfactant ratios of 1:25 and 1:100, determined using CD spectroscopy. Bottom shows α-helical content in dark gray; second from bottom shows β-sheet in light grey; third from bottom shows turns in middle grey; random coil structures are shown in dark grey at the top.

Fig. 2 shows the secondary structures of DIM-PEP-318 (A) and R-DIM-P-PEP-322 (B) in the absence and presence of SDS and DPC at peptide to surfactant ratios of 1:25 and 1:100, determined using CD spectroscopy. Bottom shows α-helical content in dark gray; second from bottom shows β-sheet in light grey; third from bottom shows turns in middle grey; random coil structures are shown in dark grey at the top.

Fig. 3 shows peptide toxicity-PI-uptake of cancer and non-cancer cell lines: (A) Concentration dependent cytotoxic activity of PEP-322 (●) and R-DIM-P-PEP-322 (▼) against melanoma cell line SBcl-2 after 8 hours of incubation with peptides; (B) Concentration dependent cytotoxic activity against primary cultures of differentiated non-tumorigenic melanocytes after 8 hour of incubation with peptides; (C) specificity of peptides at 20 µM peptide concentration after 8h of incubation displayed as PI-uptake ratio of SBcl-2 vs. melanocytes and WM164 vs. melanocytes.

Fig. 4 shows PI-uptake of various cancerous and non-cancerous cell lines upon incubation with 20µM peptide: Time dependent cytotoxic activity of PEP-322 (●), R-DIM-P-PEP-322 (▼), PEP-318 (Δ) and DIM-PEP-318 (■) against melanoma cell line SBcl-2 (A), melanoma metastasis WM164 (B), Rhabdomyosarcoma cell line TE671 (C), differentiated non-tumorigenic melanocytes cell lines (D) and normal human dermal fibroblast cell line NHDF (E) at 20 µM peptide concentration is shown.

Fig. 5 shows cytotoxicity of peptides after 8 h of incubation against SBcl-2 melanoma cell line, WM164 melanoma metastasis, TE671 rhabdomyosarcoma cell line, differentiated non-tumorigenic melanocyte cell line and NHDF normal human dermal fibroblast cell line.

Fig. 6 shows cytotoxic activity of PEP-322 (A), R-DIM-P-PEP-322 (B) and DIM-PEP-318 (C) determined by MTS cell proliferation assay against melanoma cell line SBcl-2 melanoma metastasis WM164 and non-cancer human dermal fibroblasts (NHDF). Cells were kept in the appropriate medium during incubation time of 24 h.

Fig. 7 shows spectrofluorimetric analysis of Caspase 3/7-activity of melanoma cell line SBcl-2 upon incubation of 4h with different concentrations (5µM, 10µM, 20µM, 40µM, 80µM) of peptide R-DIM-P-PEP-322 (black) and DIM-PEP-318 (white).

Fig. 8 to 11 show cytotoxicity of peptides after 1 h, 2 h, 4 h and 8 h of incubation, respectively, against SBcl-2 melanoma cell line, WM164 melanoma metastasis, TE671 rhabdomyosarcoma cell line, differentiated non-tumorigenic melanocyte cell line and NHDF normal human dermal fibroblast cell line.

Fig. 12 shows cytotoxicity of specific peptides R-DIM-P-PEP-322 and R-DIM-PEP-316 and less specific peptides R-DIM-PEP-337 and DIM-PEP-318 after 8 h of incubation, respectively, against U87 mg glioblastoma cell line, A375 melanoma cell line and NHDF-c normal human dermal fibroblast cell line and PEP-FOLD secondary structure predictions of peptides.

Fig. 13 shows secondary structure predictions of the peptides PEP-322 (A), R-DIM-P-PEP-322 (B), R-DIM-P-PEP-334 (C), R-DIM-PEP-316 (D), R-DIM-PEP-337 (E), DIM-PEP-324 (F) and DIM-318 (G), analyzed by the program PEP-FOLD.

EXAMPLES:

Experimental procedures

Materials and peptide synthesis

[0053] 1,2-Dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC), 1-Hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (POPC), 1,2-Dihexadecanoyl-sn-glycero-3-phospho-L-serine (Na-salt) (DPPS) and 1-Hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phospho-L-serine (Na-salt) (POPS) were purchased from Avanti Polar Lipids, Inc. (USA), and used without further purification. Stock solutions of DPPC and POPC were prepared in CHCl₃/CH₃OH (2:1, v/v), stock solutions of DPPS and POPS were prepared in CHCl₃/CH₃OH (9:1, v/v) and stored at -18°C.
The amidated peptides PEP-322 (PFWRIRIRR-NH₂, M=1298.6 g/mole, DIM-PEP-322 (2580.2), M= g/mole, R-DIM-PEP-322 (2580.2), M= g/mole, R-DIM-P-PEP-322 (PFWRIRIRRPRRIRIRWFP-NH₂, M= 2677.4 g/mole), PEP-318 (FWQRRIRRWRR-NH₂, M= 1715.0 g/mol) and its dimer DIM-PEP-318 (FWQRRIRRWRRFWQRRIRRWRR-NH₂, M= 3413.1 g/mol), PEP-324 (PFFWRIRIRR-NH₂, M= 1445.8 g/mol), DIM-PEP-324 (PFFWRIRIRRPFFWRIRIRR-NH₂, M= 2874.6 g/mol), PEP-316 (RWKRINRQWF-NH₂, M= 1488.8 g/mol) and R-DIM-PEP-316 (RWKRINRQWFFWQRNIRKWR-NH₂, M= 2960.5 g/mol) were purchased from NeoMPS, Inc. (San Diego, CA, USA). The purities were >96% as determined by RP-HPLC. Peptides were dissolved in acetic acid (0.1%, v/v) at a concentration of 3 mg/ml. Peptide solutions were stored at 4°C and concentrations were determined photometrically at 280 nm.

[0054] ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt) and DPX (p-xylene-bis-pyridinium bromide) used for permeability studies were purchased from Molecular Probes (Eugene, OR).

[0055] The peptides of the present invention, which have been used in part in the present example are indicated in table 1:

Table 1: List of monomeric parent peptides; the present invention includes all sequences in dimer (DIM-) (monomeric sequence + monomeric sequence) dimer retro (R-DIM-) (monomeric sequence + retro sequence), dimer with linker (DIM-X-) (monomeric sequence + X + monomeric sequence) dimer retro with linker X (R-DIM-X-) (monomeric sequence + X + retro sequence) (X= (Pro and/or, preferably or, Gly)_1-3, whereby X is preferably a single proline residue); the present invention includes as well trimer and tetrameric derivatives termed as polymeric according to dimer variations; - represents a peptide bond between the peptides and between the peptides and the linker; "retro sequence" or "reverse sequence" refers to an amino acid sequence comprising a sequence which has a reverse order than the amino acid sequence from which it is derived from (e.g. the retro sequence of ABCDE is EDCBA).

Designation	Sequence	SEQ ID No.
PEP (parent)	FQWQRNIRKVR	87
DIM-PEP	FQWQRNIRKVR - FQWQRNIRKVR	88
DIM-X-PEP	FQWQRNIRKVR X FQWQRNIRKVR	89
R-DIM-PEP	FQWQRNIRKVR RVKRINRQWQF	90
R-DIM-X-PEP	FQWQRNIRKVR X RVKRINRQWQF	91
PEP-313	FWQRNIRIRR	4
DIM-PEP-313	FWQRNIRIRR FWQRNIRIRR	92
DIM-X-PEP-313	FWQRNIRIRR X FWQRNIRIRR	93
R-DIM-PEP-313	FWQRNIRIRR RRIRINRQWF	94
R-DIM-X-PEP-313	FWQRNIRIRR X RRIRINRQWF	95
PEP-314	RRIRINRQWF	80
DIM-PEP-314	RRIRINRQWF RRIRINRQWF	96
DIM-X-PEP-314	RRIRINRQWF X RRIRINRQWF	97
R-DIM-PEP-314	RRIRINRQWF FWQRNIRIRR	98
R-DIM-X-PEP-314	RRIRINRQWF X FWQRNIRIRR	99
PEP-315	FWQRNIRKWR	17
DIM-PEP-315	FWQRNIRKWR FWQRNIRKWR	100
DIM-X-PEP-315	FWQRNIRKWR X FWQRNIRKWR	101
R-DIM-PEP-315	FWQRNIRKWR RWKRINRQWF	102
R-DIM-X-PEP-315	FWQRNIRKWR X RWKRINRQWF	103
PEP-316	RWKRINRQWF	66
DIM-PEP-316	RWKRINRQWF RWKRINRQWF	104
DIM-X-PEP-316	RWKRINRQWF X RWKRINRQWF	105
R-DIM-PEP-316	RWKRINRQWF FWQRNIRKWR	106
R-DIM-X-PEP-316	RWKRINRQWF X FWQRNIRKWR	107
PEP-317	FWQRRIRKWR	24
DIM-PEP-317	FWQRRIRKWR FWQRRIRKWR	108
DIM-X-PEP-317	FWQRRIRKWR X FWQRRIRKWR	109
R-DIM-PEP-317	FWQRRIRKWR RWKRIRRQWF	110
R-DIM-X-PEP-317	FWQRRIRKWR X RWKRIRRQWF	111
PEP-318	FWQRRIRRWRR	112
DIM-PEP-318	FWQRRIRRWRR FWQRRIRRWRR	113
DIM-X-PEP-318	FWQRRIRRWRR X FWQRRIRRWRR	114
R-DIM-PEP-318	FWQRRIRRWRR RRWRRIRRQWF	115
R-DIM-X-PEP-318	FWQRRIRRWRR X RRWRRIRRQWF	116
PEP-319	PFWQRNIRKWR	64
DIM-PEP-319	PFWQRNIRKWR PFWQRNIRKWR	117
DIM-X-PEP-319	PFWQRNIRKWR X PFWQRNIRKWR	118
R-DIM-PEP-319	PFWQRNIRKWR RWKRINRQWFP	119
R-DIM-X-PEP-319	PFWQRNIRKWR X RWKRINRQWFP	120
PEP-320	FWRNIRKWR	47
DIM-PEP-320	FWRNIRKWR FWRNIRKWR	121
DIM-X-PEP-320	FWRNIRKWR X FWRNIRKWR	122
R-DIM-PEP-320	FWRNIRKWR RWKRINRWF	123
R-DIM-X-PEP-320	FWRNIRKWR X RWKRINRWF	124
PEP-322	PFWRIRIRR	51
DIM-PEP-322	PFWRIRIRR PFWRIRIRR	125
DIM-X-PEP-322	PFWRIRIRR X PFWRIRIRR	126
R-DIM-PEP-322	PFWRIRIRR RRIRIRWFP	127
R-DIM-X-PEP-322	PFWRIRIRR X RRIRIRWFP	128
PEP-215	FWRIRIRR	75
DIM-PEP-215	FWRIRIRR FWRIRIRR	129
DIM-X-PEP-215	FWRIRIRR X FWRIRIRR	130
R-DIM-PEP-215	FWRIRIRR RRIRIRWF	131
R-DIM-X-PEP-215	FWRIRIRR X RRIRIRWF	132
PEP-227	FWRRFWRR	43
DIM-PEP-227	FWRRFWRR FWRRFWRR	133
DIM-X-PEP-227	FWRRFWRR X FWRRFWRR	134
R-DIM-PEP-227	FWRRFWRR RRWFRRWF	135
R-DIM-X-PEP-227	FWRRFWRR X RRWFRRWF	136
PEP-323	PFWRIRIRRD	59
DIM-PEP-323	PFWRIRIRRD PFWRIRIRRD	137
DIM-X-PEP-323	PFWRIRIRRD X PFWRIRIRRD	138
R-DIM-PEP-323	PFWRIRIRRD DRRIRIRWFP	139
R-DIM-X-PEP-323	PFWRIRIRRD X DRRIRIRWFP	140
PEP-324	PFFWRIRIRR	60
DIM-PEP-324	PFFWRIRIRR PFFWRIRIRR	141
DIM-X-PEP-324	PFFWRIRIRR X PFFWRIRIRR	142
R-DIM-PEP-324	PFFWRIRIRR RRIRIRWFFP	143
R-DIM-X-PEP-324	PFFWRIRIRR X RRIRIRWFFP	144
PEP-325	PFWRQRIRR	52
DIM-PEP-325	PFWRQRIRR PFWRQRIRR	145
DIM-X-PEP-325	PFWRQRIRR X PFWRQRIRR	146
R-DIM-PEP-325	PFWRQRIRR RRIRQRWFP	147
R-DIM-X-PEP-325	PFWRQRIRR X RRIRQRWFP	148
PEP-326	PFWRRQIRR	81
DIM-PEP-326	PFWRRQIRR PFWRRQIRR	149
DIM-X-PEP-326	PFWRRQIRR X PFWRRQIRR	150
R-DIM-PEP-326	PFWRRQIRR RRIQRRWFP	151
R-DIM-X-PEP-326	PFWRRQIRR X RRIQRRWFP	152
PEP-327	PFWRARIRR	53
DIM-PEP-327	PFWRARIRR PFWRARIRR	153
DIM-X-PEP-327	PFWRARIRR X PFWRARIRR	154
R-DIM-PEP-327	PFWRARIRR RRIRARWFP	155
R-DIM-X-PEP-327	PFWRARIRR X RRIRARWFP	156
PEP-328	PFWRKRIRR	54
DIM-PEP-328	PFWRKRIRR PFWRKRIRR	157
DIM-X-PEP-328	PFWRKRIRR X PFWRKRIRR	158
R-DIM-PEP-328	PFWRKRIRR RRIRKRWFP	159
R-DIM-X-PEP-328	PFWRKRIRR X RRIRKRWFP	160
PEP-329	PFWRKRLRR	55
DIM-PEP-329	PFWRKRLRR PFWRKRLRR	161
DIM-X-PEP-329	PFWRKRLRR X PFWRKRLRR	162
R-DIM-PEP-329	PFWRKRLRR RRLRKRWFP	163
R-DIM-X-PEP-329	PFWRKRLRR X RRLRKRWFP	164
PEP-330	PFWRKKLKR	82
DIM-PEP-330	PFWRKKLKR PFWRKKLKR	165
DIM-X-PEP-330	PFWRKKLKR X PFWRKKLKR	166
R-DIM-PEP-330	PFWRKKLKR RKLKKRWFP	167
R-DIM-X-PEP-330	PFWRKKLKR X RKLKKRWFP	168
PEP-331	PFWRKRWRR	56
DIM-PEP-331	PFWRKRWRR PFWRKRWRR	169
DIM-X-PEP-331	PFWRKRWRR X PFWRKRWRR	170
R-DIM-PEP-331	PFWRKRWRR RRWRKRWFP	171
R-DIM-X-PEP-331	PFWRKRWRR X RRWRKRWFP	172
PEP-332	PFWRRRIRR	57
DIM-PEP-332	PFWRRRIRR PFWRRRIRR	173
DIM-X-PEP-332	PFWRRRIRR X PFWRRRIRR	174
R-DIM-PEP-332	PFWRRRIRR RRIRRRWFP	175
R-DIM-X-PEP-332	PFWRRRIRR X RRIRRRWFP	176
PEP-333	PFWRRRWRR	58
DIM-PEP-333	PFWRRRWRR PFWRRRWRR	177
DIM-X-PEP-333	PFWRRRWRR X PFWRRRWRR	178
R-DIM-PEP-333	PFWRRRWRR RRWRRRWFP	179
R-DIM-X-PEP-333	PFWRRRWRR X RRWRRRWFP	180
PEP-334	PWRIRIRR	61
DIM-PEP-334	PWRIRIRR PWRIRIRR	181
DIM-X-PEP-334	PWRIRIRR X PWRIRIRR	182
R-DIM-PEP-334	PWRIRIRR RRIRIRWP	183
R-DIM-X-PEP-334	PWRIRIRR X RRIRIRWP	184
PEP-335	PWRRIRR	83
DIM-PEP-335	PWRRIRR PWRRIRR	185
DIM-X-PEP-335	PWRRIRR X PWRRIRR	186
R-DIM-PEP-335	PWRRIRR RRIRRWP	187
R-DIM-X-PEP-335	PWRRIRR X RRIRRWP	188
PEP-336	PWRRKIRR	84
DIM-PEP-336	PWRRKIRR PWRRKIRR	189
DIM-X-PEP-336	PWRRKIRR X PWRRKIRR	190
R-DIM-PEP-336	PWRRKIRR RRIKRRWP	191
R-DIM-X-PEP-336	PWRRKIRR X RRIKRRWP	192
PEP-337	PFWRRRIRIRR	193
DIM-PEP-337	PFWRRRIRIRR PFWRRRIRIRR	194
DIM-X-PEP-337	PFWRRRIRIRR X PFWRRRIRIRR	195
R-DIM-PEP-337	PFWRRRIRIRR RRIRIRRRWFP	196
R-DIM-X-PEP-337	RRIRIRRRWFP X RRIRIRRRWFP	197
PEP-338	RRWFFWRR	198
DIM-PEP-338	RRWFFWRR RRWFFWRR	199
DIM-X-PEP-338	RRWFFWRR X RRWFFWRR	200
R-DIM-PEP-338	RRWFFWRR RRWFFWRR	199
R-DIM-X-PEP-338	RRWFFWRR X RRWFFWRR	200
PEP-339	RRWFWRR	86
DIM-PEP-339	RRWFWRR RRWFWRR	201
DIM-X-PEP-339	RRWFWRR X RRWFWRR	202
R-DIM-PEP-339	RRWFWRR RRWFWRR	201
R-DIM-X-PEP-339	RRWFWRR X RRWFWRR	202

Preparation of liposomes

[0056] Appropriate amounts of respective phospholipid stock solution were dried under a stream of nitrogen and stored in vacuum overnight to completely remove organic solvents. The dry lipid film was then dispersed in phosphate buffered saline (PBS, 20 mM NaPi, 130 mM NaCl, pH 7.4) and hydrated at a temperature well above the gel to fluid phase transition of the respective phospholipid under intermittent vigorous vortex-mixing. The lipid concentration was 0.1 weight% for calorimetric experiments. Hydration was carried out in presence or absence of peptides at a lipid to peptide ratio of 25:1 and 12.5:1 using a protocol described for POPS (Jimenez-Monreal, A. M. et al. Biochim Biophys Acta 1373(1998), 209-219), DPPS (Jing, W. et al. J Peptide Sci 11(2005), 735-743) and DPPC (Sevcsik, E. et al. Biochim Biophys Acta 1768 (2007) 2586-2596). The fully hydrated samples were stored at room temperature until measurement.

Differential scanning Calorimetry (DSC)

[0057] DSC experiments were performed with a differential scanning calorimeter (VP-DSC) from MicroCal, Inc. (USA). Heating scans were performed at a scan rate of 30°C/h (pre-scan thermostating 30 min) with a final temperature of approximately 10°C above the main transition temperature (T_m) and cooling scans at the same scan rate (pre-scan thermostating 1 min) with a final temperature approximately 20°C below T_m. The heating/cooling cycle was performed three times. Enthalpies were calculated by integration of the peak areas after normalization to phospholipid concentration and baseline adjustment using the MicroCal Origin software (VP-DSC version). The phase transition temperature was defined as the temperature at the peak maximum (McElhaney, R. N. Chem Phys Lipids 30(1982), 229-259).

Circular Dichroism Spectroscopy

[0058] Measurements were performed on a Jasco J 715 Spectropolarimeter (Jasco, Germany) at room temperature using quartz cuvettes with an optical path length of 0.02 cm. The CD spectra were measured between 260 nm and 180 nm with a 0.2 nm step resolution. To improve accuracy 5 scans were averaged. Peptides were dissolved in 10 mM Hepes (pH 7.4) to a final concentration of 100 µM. Spectra were measured in the absence and presence of 1 mM sodium dodecyl sulfate (SDS) and 1 mM dodecylphosphocholine (DPC) mimicking cancer and healthy mammalian membranes, respectively. The respective peptide to surfactant molar ratios were 1:25 and 1:100. Background signals were abstracted after measurements. Percentage secondary structure calculations were done using Dichroweb, CDSSR Convolution Program using reference set 4 (Whitmore, L. and Wallace, B. A. Biopolymers 89(2008), 392-400 and Nucleic Acids Res. 32(2004), W668-W673.

Fluorescence spectroscopy

[0059] Fluorescence spectroscopy experiments were performed using a SPEX Fluoro Max-3 spectrofluorimeter (Jobin-Yvon, France) and spectra were analyzed with Datamax software.

ANTS/DPX leakage

[0060] Leakage of aqueous contents from liposomes was determined using the 8-aminonaphthalene-1,3,6-trisulfonic acid/p-xylene-bis-pyridinium bromide (ANTS/DPX) assay. Lipid films were hydrated with 12.5 mM ANTS, 45 mM DPX, 68 mM NaCl, 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) at pH 7.4 following a standard procedure.

[0061] Subsequently, the dispersions were extruded 20 times through a polycarbonate filter of 0.1 µm pore size to obtain LUVs. Unilamellarity and size were tested by X-ray and dynamic light scattering, respectively. The ANTS/DPX encapsulating vesicles were separated from free ANTS/DPX by exclusion chromatography using a column filled with Sephadex G-75 (Amersham Biosciences) fine gel swollen in an iso-smotic buffer (10 mM HEPES, 140 mM NaCl, pH 7.4). The void volume fractions were collected and the phospholipid concentration was determined by phosphate analysis (Broekhuyse, R. M. Biochim. Biophys. Acta 152(2005), 307-315; Tao, T. and Cho, J. Biochemistry 18(1979), 2759-2765).

[0062] The fluorescence measurements were performed in 2 mL of the isosmotic buffer in a quartz cuvette at room temperature. Aliquots of LUVs were diluted with the iso-osmotic buffer to a final lipid concentration of 50 µM. Fluorescence spectra were obtained at 37°C using an excitation wavelength of 360 nm and an emission wavelength of 530 nm and a slit width of 5 nm for both excitation and emission monochromators. Fluorescence emission was recorded as a function of time before and after the addition of incremental amounts of peptide. The fluorescence increase due to leakage and subsequent dilution of quenched dye was measured after addition of peptides. Peptides were added to final concentrations of 2, 4 and 8 µM, corresponding to peptide to lipid molar ratios of 1:25, 1:12.5 and 1:6.25, respectively.

[0063] Data are presented in terms of fluorescence intensity (IF):

[0064] F is the measured fluorescence, F₀ the initial fluorescence without peptide and F_max the fluorescence corresponding to 100% leakage gained by addition of 1% Triton X-100.

Tryptophan quenching

[0065] Tryptophan fluorescence spectra were obtained at room temperature using an excitation wavelength of 282 nm and a slit width of 5 nm for excitation and emission monochromators. Quenching of Tryptophan was carried out in the presence and absence of phospholipid liposomes (lipid to peptide ratio 25:1) using 0.1, 0.4 and 0.7 M acrylamide. The data were analyzed according to the Stern-Volmer equation:


where F₀ and F represent the fluorescence emission intensities in the absence and presence of the quencher molecule (Q) and K_SV is the Stern-Volmer quenching constant, which is a quantitative measure for the accessibility of tryptophan to acrylamide (Tao, T. and Cho, J. Biochemistry 18(1979), 2759-2765).

Cell lines and Culture

[0066] The primary human melanoma cell line SBcl2 and the metastatic melanoma WM164 were maintained in RPMI (Sigma) supplemented with 2% FBS, 2% L-glutamine and 1% Pen/Strep. Glioblastoma (U87-mg) purchased from CLS (Cell Line Service Heidelberg, Germany) and Rhabdomyosarcoma cell lines (TE671) purchased from ECAAC (Health Protection Agency Culture Collections Salisbury, UK) are cultured in Dulbecco's Modified Eagle Medium (DMEM) with addition of 2 mM Glutamine, 10% FBS (fetal bovine serum). Human melanocytes used as healthy control cells: were isolated from the foreskin. The foreskin was cut into small pieces and incubated with 0.3% trypsin (PAA) overnight at 4°C and for one hour at 37°C. Epidermis was separated. Cells were mechanically removed from the cell layer and centrifuged at 300g for 3 min. The pellet was resuspended in melanocyte growth media (Biomedica). Melanocytes were further cultured in human melanocytes growth medium (PromoCell GmbH). Normal human dermal fibroblasts purchased from (NHDF) (PromoCell GmbH) were cultured in fibroblast growth medium 2 (PromoCell GmbH). All cells were kept in a 5% CO₂ atmosphere at 37°C. At 90% confluency cell-culture flasks were passaged with accutase. All cell cultures were periodically checked for mycoplasma.

PI-uptake assay

[0067] For detection of PI-uptake by fluorescence spectroscopy experiments were performed according to the following protocol.

[0068] Cells were collected, resuspended in media and diluted to a concentration of 10⁶ cells/ml. Aliquots of 10⁵ cells were incubated with peptides for up to 8 hours at 37°C and 5% CO₂. PI was added and cells were again incubated for 5 min at room temperature in the dark. Excitation and emission wavelengths were 536 nm and 617 nm, respectively.

[0069] Cytotoxicity was calculated from the percentage of PI positive cells in media alone (P₀) and in the presence of peptide (P_X). Triton-X was used to determine 100% of PI positive cells (P₁₀₀).

[0070] For detection of PI-uptake by fluorescence microscopy experiments were performed on a Leica DMI6000 B with IMC in connection with a Leica DFC360 FX camera and AF 6000 software.

[0071] Cells (1-5x10⁴) were seeded on Ibidi µ-Slide 8 wells and grown in 300µl media for 2-3 days to a confluent layer. Propidium iodide (PI, 2 µl of 50 µg/ml in PBS) was added to the well and cell status was checked after 5 min of incubation in the dark at room temperature. Then, peptides were added to the desired concentration and peptide effect was followed immediately. Pictures were taken every 5 or 15 min for up to 8 h from the same section of cells. Excitation and emission wavelength were as follows: PI excitation, 535 nm and emission, 617 nm.

MTS viability assay

[0072] Cell proliferation was measured by using a CellTiter 96 AQ nonradioactive cell proliferation assay (Promega). Cells were plated in 96-well plates and grown until confluency. Peptides were added to a final concentration of 5-100µM. After incubation for 24 h at 37°C (5 % CO₂) MTS [3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]-phenozine methosulfate solution (20 µl/well) was added and cells were again incubated for 2 h at 37°C (5 % CO₂). The MTS compound is bioreduced by cells into a colored formazan product that is soluble in tissue culture medium. The quantity of the formazan product as measured by the amount of 490nm absorbance is directly proportional to the number of living cells in culture. Data are calculated as a percentage of the control (untreated) samples and represent the average of three wells in one experiment which was repeated three times per cell line.

Spectrofluorimetric analysis of caspase-3/7 activity

[0073] 5 x 10⁵ cells / ml were seeded into 96-well plate and grown overnight at 37°C and 5% CO₂. Cells were incubated with different concentrations of peptide for 4 hours. Apo-ONE® caspase-3/7 reagent was added in a 1:1 volume ratio and cells were incubated for 4 hours. Cells were then analyzed by fluorescence spectroscopy (GloMax®-Multi+ Microplate Multimode Reader with Instinct™). Untreated cells were used as negative control. Analysis was performed with Apo-ONE® Homogeneous Caspase-3/7 Assay (see Fig. 7).

Hemolysis

[0074] The hemolytic activity towards human red blood cells (RBCs), which were obtained from heparinized human blood, was determined by the release of hemoglobin following one hour incubation at 37°C in MHNA (Mueller Hinton cation Non Adjusted). Percentage of hemolysis of RBCs was calculated using 1 % Triton as 100 % lysis and PBS as 0 % lysis, peptide concentration was 500µg/ml.

Results

[0075] In the present examples, toxicity, respectively selectivity of the peptides of the present invention against melanoma, rhabdomyosarcoma and glioblastoma cell lines that expose the negatively charged lipid phosphatidylserine on the outside was examined. Selective peptides are not toxic against normal non-tumor cells as melanocytes and fibroblasts or red blood cells in the same concentration range.

[0076] Selective and active peptides can be (retro-) dimers of the peptides of the present invention with or without linkers exhibiting defined secondary structures (as defined above) and show cancer selective activity in vitro and in cancer model systems.

[0077] Exemplarily 4 peptides were chosen to represent the observed effects. The results present data gained on peptide PEP-322 and PEP-318, representative for monomeric but non-active peptides, R-DIM-P-PEP-322 representative for cancer active and specific peptides and DIM-PEP-318 representative for cancer and non-cancer active, non-selective peptides (see Table 2).

[0078] Data gained on further peptides studied are reported after the result part. Besides R-DIM-P-PEP-322 the peptides, DIM-PEP-322, R-DIM-PEP-322, R-DIM-PEP-316, R-DIM-PEP-323, DIM-PEP-324, R-DIM-P-PEP-324, R-DIM-P-PEP-332, R-DIM-P-PEP-334 were shown to be selective for cancer cells.

Table 2: Overview of peptide sequences, net charge and hydrophobicity of the peptides examined

	Sequence	Net charge	ΔGwifa [kcal/mol]
PEP-322	PFWRIRIRR	+5	n.d.
R-DIM-P-PEP-322	PFWRIRIRRPRRIRIRWFP	+9	-2.00/-2.60
PEP-318	FWQRRIRRWRR	+7	n.d.
DIM-PEP-318	FWQRRIRRWRRFWQRRIRRWRR	+13	-8.17/-7.91

a Peptide hydrophobicity expressed as transfer free energy of peptides from water to bilayer interface (ΔGwif) calculated from the whole-residue hydrophobicity scale taking into account the contribution of the C-terminal amide (48). As % helix the respective CD data for SDS/DPC were taken. Since for the short peptides PEP-322 and -318 the CD measurements are not accurate enough the ΔGwif was only calculated for the dimer peptides.

Peptide structure - activity and selectivity

In silico - Secondary Structure Prediction

[0079] "Dimers" of several PEP peptides were first analyzed by simulation of the secondary structure. The secondary structure of putative membrane active dimer peptides were predicted by the online program PEP-FOLD: http://bioserv.rpbs.univ-parisdiderot.fr/PEP-FOLD/ (Maupetit, J et al Nucleic Acids Res. 37(2009), W498-W503). From this analysis several peptides were selected for synthesis and activity studies according to their high proportion of amphipathic β-sheet or α-helical structure.

[0080] The non-active monomeric peptides turned out to be too short for assembly of a defined secondary structure, however assuming formation of an α-helix, it is only amphiphatic in the case of PEP-318. Interestingly the cancer specific peptides (DIM-, R-DIM-, R-DIM-P- PEP-322, DIM-PEP-324 and R-DIM-PEP-316 formed 2 β-strands or 2 α-helices (R-DIM-PEP-316) with a turn in the middle and distribution of cationic and hydrophobic regions. For the active but non-selective peptide DIM-PEP-318 a linear amphipathic α-helix without a loop was predicted (see Table 3, Fig. 13).

Table 3: List of monomeric parent peptides; list includes all sequences in dimer (DIM-) (monomeric sequence + monomeric sequence as defined herein), dimer retro (R-DIM-) (monomeric sequence + retro sequence), dimer with linker X (DIM-X-) (monomeric sequence + X + monomeric sequence), dimer retro with linker X (R-DIM-X-) (monomeric sequence + X + retro sequence). H = helix, T = turn, β = β-strand. For positive or negative peptide specificity for tumor over non tumor cells in the case of studied peptides the respective -fold specificity for melanoma (SBcl2 or A375) over non tumor skin cells (melanocytes or normal human dermal fibroblasts (NHDF-c)) at 20µM peptide concentration after 8 hours incubation is listed (derived by PI uptake studies).

monomeric peptide parents	Secondary Structure					Specificity
	Mon**	DIM-	DIM-X-	R-DIM-	R-DIM-X-
PEP	H	H	2H T	H	2H T
PEP-313		H T	2β T	H	H T β
PEP-314	H	2H	2β T	3β 2T	2β T
PEP-315	H	H	2H T	H	2H T
PEP-316	H	H T	2H T	2H T	2H T	Yes (15fold) (R-DIM-)
PEP-317	H	H	2H T	H	2H T
PEP-318	H	H	2H T	H	2H T	No (<1fold) (DIM -)
PEP-319	H	2H T	2H T	H	2H T
PEP-320	H	H	2H T	2H T	2H T
PEP-322		2β T	2β T	2β T	2β T	yes (50fold, 20fold, >100 fold) (DIM-, R-DIM-, R-DIM-X-)
PEP-215		2β T	2β T	2β T	2β T
PEP-227		2H	2H T	H	2H T
PEP-323		2β T	2β T	2β T	4β T
PEP-324	H	2β T	2β T	2β T	2β T	yes (7fold) (DIM-), (15fold) (R-DIM-X-)
PEP-325	H	2H T	2H T	H	2H T
PEP-326	H	2H T	2H T	H	2H T
PEP-327	H	2H T	2H T	H	2H T
PEP-328	H	2H	2H T	H	2H T
PEP-329	H	2H	2H T	H	2H T
PEP-330	H	2H	2H T	H	2H T
PEP-331	H	2H	2H T	H	2H T
PEP-332	H	2H	2H T	H	2H T	(5fold) (R-DIM-X-)
PEP-333	H	2H	2H T	H	2 H T
PEP-334		2β T	2β T	2β T	2β T	(15fold) (R-DIM-X-)
PEP-335		2H	2H T	H	2H T
PEP-336		H	2H T	H	2H T
PEP-337	H	2H T	2H T	H	2H T	weak (2fold) (R-DIM-)
PEP-338		2β T	2β T	= DIM-	=DIM-X-
PEP-339		2β T	2β T	= DIM-	=DIM-X-

**partially no secondary structure predictable due to low number of amino acids

Circular dichroism spectroscopy - secondary structure vs. activity and selectivity

[0081] Strikingly the selective peptide R-DIM-P-PEP-322 (Figure 1B) exhibits a significant increase of β-sheet conformation in the presence of the cancer mimic SDS, α-helical content is even further decreased. Moreover the structure of the peptide in the presence of the healthy mimic DPC is the same as in solution, giving further hint for the cancer selective toxicity of these peptides (see also Figure 2).

[0082] Percentage secondary structure calculations were done using Dichroweb, CDSSR Convolution Program using reference set 4 (40;50). The α-helical content is shown in dark gray at the bottom; β-turns in light grey; turns in middle grey; random coil structures in dark grey at the top.

[0083] Fig. 2 now presents the results of circular dichroism spectroscopy of the non-specific peptide DIM-PEP-318 in contrast to the specific peptide R-DIM-PEP-322. In contrast to the PEP-322 peptide group, DIM-PEP-318 possesses a higher α-helical content in the presence of the cancer mimic SDS as well as in the presence of the non-cancer mimic DPC. DIM-PEP-318 adopts up to 75 % α-helical structure without discrimination between cancer and non-cancer cell mimic.

[0084] Figure 2: Secondary structures of DIM-PEP-318 (A) and R-DIM-P-PEP-322 (B) in the absence and presence of SDS and DPC at peptide to surfactant ratios of 1:25 and 1:100, determined using CD spectroscopy. The α-helical content is shown in dark gray at the bottom; β-turns are shown in light grey; turns are shown in middle grey; random coil structures are shown in dark grey at the top.

Model studies

In vitro studies - membrane permeabilization

PI-uptake - increase of activity by sequence doubling

[0085] Cytotoxic activity of the peptides towards melanoma cells of primary (SBcl-2) and metastatic lesions (WM164) and differentiated non-tumorigenic melanocytes was determined by measurement of PI-uptake, which only occurs when integrity of the cell membrane is lost. Cells were incubated in media containing serum for 8h in the presence of peptides. Peptide concentrations were varied from 10 to 80µM. Figure 3 illustrates that the monomer PEP-322 is only minor active against the melanoma cell line SBcl-2 with less than 5% killing at a peptide concentration of 80µM, as well as against melanocytes with a moderate two-fold selectivity for WM164 cells at 20µM peptide concentration (Fig. 3C). The dimeric peptide R-DIM-P-PEP-322 shows strongly increased activity against SBcl-2 compared to the monomer with very high specificity for the melanoma cell line. Already at a peptide concentrations of 20 µM, R-DIM-P-PEP-322 yields more than 80% PI positive SBcl-2 cells, while only less than 1% of differentiated non-tumorigenic melanocytes are killed (Fig. 3B), exhibiting a specificity more than 100-fold for cancer cells (see Fig. 3C). The second melanoma cell line, WM164, tested at 20µM R-DIM-P-PEP-322 peptide concentration is also highly sensitive for the peptide.

PI-uptake - specificity and time dependence of killing

[0086] Cytotoxic activity of the peptides towards melanoma cells of primary (SBcl-2) and metastatic lesions (WM164), a rhabdomyosarcoma cell line (TE671) and their healthy counterparts differentiated non-tumorigenic melanocytes and normal human dermal fibroblasts (NHDF) was determined by measurement of PI-uptake, which indicates a loss of cell membrane integrity (Fig. 4 and 5). Cells were incubated in media containing serum for up to 8h in the presence of peptides at 20 µM peptide concentration.

[0087] Both monomeric short peptides PEP-322 as well as PEP-318 are only minor active against cancer cells (< 30%, see Figure 4A-C). However, PEP-318 is even slightly active against non-cancer cells, killing up to ~30% of HNDF. Interestingly, the dimer R-DIM-P-PEP-322 shows high cancer toxicity (up to 80%) but with negligible non-cancer toxicity (see Figure 4D-E). In contrast to DIM-PEP-318, R-DIM-P-PEP-322 kills quite slowly reaching its highest activity not before 4-8h. The second dimer, namely DIM-PEP-318, possesses the highest and fastest anticancer activity with up to 90 % killing within minutes (Fig.4A-C). However, DIM-PEP-318 reveals to be quite unspecific since it is also highly active against differentiated non-tumorigenic melanocytes as well as against normal human dermal fibroblasts (Fig. 4D-E). Cytotoxic activity of the peptides after 8 h of incubation is given in Figure 11.

Cell viability - MTS cell proliferation

[0088] To determine long-time toxicity of peptides, a MTS cell proliferation assay was used to elucidate cell viability upon 24 h incubation with variant peptide concentration and human melanoma cell lines SBcl-2 and WM164 and non-differentiated human skin fibroblast cell line NHDF. As shown in Fig. 6A PEP-322 affects cancer cells and non- cancer cells only marginally even at higher peptide concentrations of 100 µM sustaining more than 70% cell viability (IC_{50 WM164 and NHDF} > 100 µM) and 40% (IC_{50 SBcl-2} 90). Dimerization could highly improve anticancer activity. R-DIM-P-PEP-322 exhibits a decreased IC₅₀ value of 8 µM and 15 µM for melanoma cell line SBcl-2 and melanoma metastasis WM164, respectively (Fig. 6B) compared to an IC₅₀ value of 80 µM for the non-cancer cell line, yielding 8-5-fold selectivity for cancer cells. DIM-PEP-318 shows also high activity against cancer cells resulting in an IC₅₀ < 10 µM (Fig. 6C). As indicated already by PI uptake studies this peptide is as toxic for non-cancer cells, revealed by a very low IC₅₀ of 10µM for NHDF, as well. Results of MTS and PI assay with cancer cells correlated well. An overview of IC₅₀ values is given in Table 6.

Table 6: Comparison of IC₅₀ values determined through PI-uptake (8 h) and MTS cell viability assay (24 h).

	SBcl-2 (PI / MTS)	Fibroblasts (PI / MTS)
PEP-322	>80 µM / 90 µM	>>80 µM / >100 µM
R-DIM-P-PEP-322	8 µM / 8 µM	>>80 µM / 80µM
PEP-318	n.d. / n.d.	n.d. / n.d.
DIM-PEP-318	<20 µM / 6 µM	<20 µM / 10 µM

Hemolytic activity against red blood cells - specificity

[0089] Hemolytic activity of peptides against red blood cells was tested at 500 µg/ml peptide and 2.5% red blood cell concentration. Very interestingly, only an increase in the hydrophobic content by N-acylation, 6-MO-PEP-322, lead to a slightly elevated hemolytic activity of approximately 10-fold cell lysis compared to non-acylated peptides (see Table 7). Further it was very surprising that DIM-PEP-318 was not hemolytic, considering the high toxicity towards melanocytes and fibroblasts.

Table 7: Hemolytic activity of peptides against human red blood cells

	% lysis of 2.5% RBCsa	IC50 [µg/ml]
PEP-322	2b	>500b
R-DIM-P-PEP-322	0.84+ 0.63	>500
PEP-318	n.d.	n.d.
DIM-PEP-318	2.87+ 0.57	>500

a Percentage of hemolysis of human red blood cells (RBCs) was calculated following one hour incubation at 37°C in PBS using 1% Triton as 100% lysis and PBS as 0% lysis, peptide concentration was 500µg/ml. b from Zweytick et al., 2011 (55). n.d. not determined

Caspase-3 cleavage-apoptosis or necrosis

[0090] To clearly differentiate between necrotic and apoptotic killing a caspase-3/7 activity assay was used to detect emergence of apoptosis (Fig. 7). R-DIM-P-PEP-322 showed apoptosis indicated by a strong increase of caspase-3/7 activity from the time-point of 4 hours of incubation of melanoma cells SBcl-2 with 10-20µM peptide, indicated by a 200-fold increase of green fluorescence. The non-specific peptide DIM-PEP-318 showed much lower caspase-3/7 activity, indicating a non-apoptotic killing mechanism like necrosis shown by a strong PI-uptake by the peptide (Figure 8-11).

[0091] Additionally, apoptotic like blebbing of the cell membrane is observed during incubation of the rhabdomyosarcoma cell line TE671 in the presence of the peptide.

Table 8: Correlation of activity exhibited by peptides PEP-322 and R-DIM-P-PEP-322 in model and in vitro studies.

Peptide	PEP-322	R-DIM-P-PEP-322
amino acid sequence	PFWRIRIRR-(NH2)	- PRRIRIRWFP-NH2
net charge	+5	+9

cancer mimic /healthy mimic
bilayer perturbation -DSC	+ / -	+++ / -
permeability -ANTS/DPX leakage	+ / -	+++ / -
bilayer affinity -quenching	+ + / -	++ / -
structure - CD	> β-sheet/ as in solution	> β-sheet/ as in solution

cancer cells / healthy cells
toxicity - PI uptake, MTT	- / -	+++ / -
cancer specificity	(+)	++++

Table 9: Correlation of activity exhibited by selective peptide R-DIM-P-PEP-322 and non-selective peptide DIM-PEP-318 in model and in vitro studies.
Peptide	R-DIM-P-PEP-322	DIM-PEP-318
amino acid sequence	PFWRIRIRR-P-RRIRIRWFP-NH2	FWQRRIRRWRR-FWQRRIRRWRR-NH2
net charge	+9	+13
cancer mimic /healthy mimic
bilayer perturbation - DSC	+++ / -	+++ / +++
permeability - leakage	+++ / -	+++ / +
bilayer affinity -quenching	++ / -	++ / +
structure - CD	> β-sheets / as in solution	> α-helical / > α-helical

cancer cells / healthy cells
toxicity - PI up-take, MTT	+++ / -	+++ / +++
cancer specificity	++++	---

Discussion

[0092] In this example the selective antitumor activity of the peptides of the present invention could be demonstrated. The monomeric PEP-322 exhibited only weak activity against melanoma cancer cell lines, the dimeric form R-DIM-P-PEP-322 (comprising 2 beta-strands separated by a turn) showed highly increased activity. PI-uptake of melanoma cells upon incubation with peptide R-DIM-P-PEP-322 further demonstrates that the peptide operates via a membrane mediated way, since PI can only be taken up by cells that suffer membrane disintegration. Improved interaction of the dimer with the cancer mimic PS correlated with increased activity against the melanoma cancer cell line and non-interaction with the healthy mimic PC correlated with non-toxicity against non-cancer melanocytes. The dimer exhibits a high membrane destabilization emphasized by highly increased membrane permeability of PS bilayers. Besides, permeability studies show that a certain threshold concentration of the dimer is needed for induction of sufficient leakage of ANTS/DPX, differentiating it from highly lytic but mostly unspecific peptides like melittin. In agreement also the effect on neutral lipids is negligible. Moreover by calorimetric studies it could be demonstrated that the effect of the dimer is even much higher than that of the monomer at doubled concentration, rather suggesting a structural effect than a simple mass and charge effect.

[0093] Trp localization studies of peptides showed that if a peptide is active against a certain membrane, it exhibits a significant blue shift of Trp emission wavelength upon interaction with the membrane indicating a more hydrophobic environment of Trp due to interaction with the membrane interface. In the case of the monomer PEP-322 and dimer R-DIM-P-PEP-322 the blue shift is only detected in presence of the target lipid PS present on the surface of cancer membranes, whereas in the presence of PC no blue shift appears, going hand-in-hand with a selective toxicity against cancer cells in vitro. These findings are in line with the ability of Trp quenching, which is strongly decreased only in the presence of the target lipid PS. Non-selective peptides like DIM-PEP-318 however reveal a blue shift in the presence of both model systems.

[0094] Further structural information on the studied peptides was given by CD experiments. Again structural changes for PEP-322 and the dimer appear only in the presence of the negatively charged cancer mimic (SDS). The peptide DIM-PEP-318 changes its structure in environment of both models conform able to its low specificity. Only the non-selective peptide shows an increase of the α-helical content in the presence of both model systems, differently PEP-322 and R-DIM-P-PEP-322 show an increase of the β-sheet content upon presence of the cancer model SDS.

[0095] From the differences in activity displayed by the monomer and the dimer it was however surprising that both peptides show quite similar structural characteristics in solution and model system. Considering the shortness of PEP-322, it is even questionable if a β-sheet conformation is possible. It is moreover reasonable that two monomer peptide stretches arrange on the lipid surface like a dimer not covalentely linked. The real dimer however is fixed in this conformation and will create stronger membrane perturbance and finally higher membrane permeabilization, which can explain its highly increased activity in model and cell system.

[0096] On the one hand it was demonstrated that high membrane interaction of the peptides derived from the membrane active peptide PEP with anionic PS correlates with high activity against melanoma cells, on the other hand it could be shown that increasing interaction with the healthy mimic neutral PC correlates with decreased specificity indicated by increased interaction with non-cancer cell types like melanocytes or fibroblasts. This was demonstrated for the dimer of PEP-318, namely DIM-PEP-318 also originally derived from PEP. The short monomeric peptides PEP-322 and PEP-318 are only minor active against cancer cells even if incubation time is extended to 8 h or higher peptide concentrations are used. Partially the low activity can also be due to less defined structure. In contrast, the dimeric peptides DIM-PEP-322 and DIM-PEP-318 exhibit increased anti-cancer activity. However the different peptides seem to operate by different mechanisms, since peptide DIM-PEP-318 reaches its maximum toxicity against cancer cells already after 15 minutes, whereas contrariwise the dimer R-DIM-PEP-322 kills much slower reaching its maximum activity not before 8 hours. Nevertheless it shows similar or even increased cancer toxicity compared to DIM-PEP-318, after long time period. The different time dependence of cell killing by the peptides indicates 2 different killing mechanism. The very fast action of DIM-PEP-318 gives strong evidence for a direct membranolytic effect causing necrosis. Prediction of the secondary structure of the peptides proposes an amphiphatic α-helix. Structural analysis through CD spectroscopy of DIM-PEP-318 also reveals induction of a mostly α-helical structure in the presence of the cancer as well as the non-cancer mimic resulting in non-selective lysis of cells. The selective peptide R-DIM-P-PEP-322 obviously acts via a different mechanism. The relatively slow action together with the observation of membrane blebbing and Caspase-3/7 activity is an indication for membrane-mediated apoptosis. For induction of apoptosis the peptide has to enter the cell specifically over probably the PS compartments on the surface and further reach another negatively charged target on the surface of cancer cell mitochondria, like cardiolipin. Successive swelling of mitochondria and release of cytochrome-C activate the caspase dependent pathway of the programmed cell death. Interestingly R-DIM-P-PEP-322 shows induction of an increase in the predominant β-sheet structure with a turn and no changes in structure in the presence of the non-cancer cell mimic. In the secondary structure prediction an arrangement of 2 β-strands with hydrophobic endings with a loop in the middle composed of cationic amino acids was predicted. According to other prediction studies the amphipathic distribution of amino acids with a loop between 2 β-strands or 2 α-helices seem to be important structural features for an active and specific peptide.

Claims

1. An isolated peptide to be used in the treatment of cancer consisting of 12 to 50 amino acid residues comprising

- at least two beta-strands, or

- at least two alpha-helices or

- at least one beta-strand and at least one alpha-helix,
wherein said beta-strands and/or alpha-helices are separated from each other by at least one turn, wherein the peptidehas a positive net charge of +7 or more.

2. Peptide for use according to claim 1, wherein the peptide comprises at least 5 hydrophobic amino acid residues.

3. Peptide for use according to claim 1 or 2, wherein the isolated peptide comprises at least one peptide having amino acid sequence (X₁)_M-X₂-(X₃)_P-X₄-(X₅)_Q-X₆-(X₇)_S or the reverse sequence thereof, wherein
X₁ is a hydrophobic amino acid, preferably selected from the group consisting of phenylalanine (Phe), alanine (Ala), leucine (Leu) and valine (Val),
X₂ is a hydrophobic amino acid, preferably tryptophan (Trp), X₃ is selected from the group consisting of alanine (Ala), arginine (Arg), glutamine (Gln), asparagine (Asn), proline (Pro), isoleucine (Ile), leucine (Leu) and valine (Val),
X₄ is selected from the group consisting of isoleucine (Ile), phenylalanine (Phe), tryptophan (Trp) and tyrosine (Tyr),
X₅ is selected from the group consisting of arginine (Arg), lysine (Lys), tyrosine (Tyr) and phenylalanine (Phe),
X₆ is a hydrophobic amino acid, preferably selected from the group consisting of isoleucine (Ile), tryptophan (Trp), valine (Val) and leucine (Leu), and
X₇ is selected from the group consisting of arginine (Arg), lysine (Lys), isoleucine (Ile) and serine (Ser), and wherein
M is 1 or 2,
Q is 1 or 2,
P is 2 or 3, and
S is 1, 2, 3 or 4 under the proviso that if (X₅)_Q is Arg-Arg S is 1.

4. Peptide for use according to claim 3, wherein the at least one peptide have an amino acid sequence selected from the group consisting of FWQRIRKVR (SEQ ID No. 1), FWQRRIRKVRR (SEQ ID No. 2), FWQRKIRKVRK (SEQ ID No. 3), FWQRNIRIRR (SEQ ID No. 4), FWQRNIRKVR (SEQ ID No. 5), FWQRNIRVR (SEQ ID No. 6), FWQRNIRKVRR (SEQ ID No. 7), FWQRNIRKVKK (SEQ ID No. 8), FWQRNIRKVRRR (SEQ ID No. 9), FWQRNIRKVKKK (SEQ ID No. 10), FWQRNIRKVRRRR (SEQ ID No. 11), FWQRNIRKVRRRI (SEQ ID No. 12), FWQRNIRKVKKKK (SEQ ID No. 13), FWQRNIRKVKKKI (SEQ ID No. 14), FWQRNIRKIR (SEQ ID No. 15), FWQRNIRKLR (SEQ ID No. 16), FWQRNIRKWR (SEQ ID No. 17), FWQRNWRKVR (SEQ ID No. 18), FWQRNFRKVR (SEQ ID No. 19), FWQRNYRKVR (SEQ ID No. 20), FWQRNIRKVS (SEQ ID No. 21), FWQRRIRIRR (SEQ ID No. 22), FWQRPIRKVR (SEQ ID No. 23), FWQRRIRKWR (SEQ ID No. 24), FWPRNIRKVR (SEQ ID No. 26), FWARNIRKVR (SEQ ID No. 27), FWIRNIRKVR (SEQ ID No. 28), FWLRNIRKVR (SEQ ID No. 29), FWVRNIRKVR (SEQ ID No. 30), FWQRNIFKVR (SEQ ID No. 31), FWQRNIYKVR (SEQ ID No. 32), FAWQRNIRKVR (SEQ ID No. 33), FLWQRNIRKVR (SEQ ID No. 35) and FVWQRNIRKVR (SEQ ID No. 36) or the reverse sequence thereof.

5. Peptide for use according to any one of claims 1 to 4, wherein the isolated peptide comprises at least one peptide having amino acid sequence (X_1')_M' - X_2' - (X_3')_P' - (X_4')_Q' - (X_5')_T' - (X_6')_R' - (X_7')_S' or the reverse sequence thereof, wherein
X_1' is a hydrophobic amino acid, preferably selected from the group consisting of phenylalanine (Phe) and isoleucine (Ile),
X_2' is a hydrophobic amino acid, preferably tryptophan (Trp),
X_3' is selected from the group consisting of glycine (Gly), asparagine (Asn), isoleucine (Ile) and phenylalanin (Phe),
X_4' is isoleucine (Ile) or tryptophan (Trp),
X_5' is arginine (Arg) or lysine (Lys),
X_6' is a hydrophobic amino acid, preferably selected from the group consisting of isoleucine (Ile), tryptophan (Trp) and valine (Val) and
X_7' is arginine (Arg), and wherein
M' is 1 or 2,
T' is 1 or 2,
R' is 0 or 1,
P' is 1, 2 or 3,
Q' is 1, and
S' is 0, 1 or 2.

6. Peptide for use according to claim 5, wherein the at least one peptide have an amino acid sequence selected from the group consisting of FWRIRKWR (SEQ ID No. 37), FWRIRKVR (SEQ ID No. 38), FWRWRR (SEQ ID No. 39), FWRRWRR (SEQ ID No. 40), FWRRWIRR (SEQ ID No. 41), FWRGWRIRR (SEQ ID No. 42), FWRRFWRR (SEQ ID No. 43), FWRWRWR (SEQ ID No. 44), FWRIWRWR (SEQ ID No. 45), FWRIWRIWR (SEQ ID No. 46), FWRNIRKWR (SEQ ID No. 47) and FWRRRIRIRR (SEQ ID No. 48) or the reverse sequence thereof.

7. Peptide for use according to any one of claims 1 to 6, wherein the isolated peptide comprises at least one peptide having amino acid sequence (X_1")_M" - X_2" - (X_3")_P" - (X_4")_Q" - (X_5")_R" - (X_6")_S" or the reverse sequence thereof, wherein
X_1" is a hydrophobic amino acid, preferably selected from the group consisting of proline (Pro) and phenylalanine (Phe),
X_2" is a hydrophobic amino acid, preferably tryptophan (Trp),
X_3" is selected from the group consisting of alanine (Ala), arginine (Arg), glutamine (Gln), lysine (Lys), tryptophan (Trp) and isoleucine (Ile),
X_4" is selected from the group consisting of arginine (Arg) and aspartate (Asp),
X_5" is a hydrophobic amino acid, preferably selected from the group consisting of isoleucine (Ile), tryptophan (Trp), phenylalanine (Phe), valine (Val) and leucine (Leu), and
X_6" is selected from the group consisting of arginine (Arg), lysine (Lys), isoleucine (Ile), serine (Ser) and aspartate (Asp), and wherein
M" is 0, 1, 2 or 3,
Q" is 0, 1, 2 or 3,
R" is 1 or 2,
P" is 1, 2 or 3, and
S" is 1, 2 or 3.

8. Peptide for use according to claim 7, wherein the at least one peptide have an amino acid sequence selected from the group consisting of PFWRWRIWR (SEQ ID No. 50), PFWRIRIRR (SEQ ID No. 51), PFWRQRIRR (SEQ ID No. 52), PFWRARIRR (SEQ ID No. 53), PFWRKRIRR (SEQ ID No. 54), PFWRKRLRR (SEQ ID No. 55), PFWRKRWRR (SEQ ID No. 56), PFWRRRIRR (SEQ ID No. 57), PFWRRRWRR (SEQ ID No. 58), PFWRIRIRRD (SEQ ID No. 59), PFFWRIRIRR (SEQ ID No. 60), PWRIRIRR (SEQ ID No. 61), PFWRRQIRR (SEQ ID No. 81), PFWRKKLKR (SEQ ID No. 82), PWRRIRR (SEQ ID No. 83), PWRRKIRR (SEQ ID No. 84) and PFWRRIRIRR (SEQ ID No. 85) or the reverse sequence thereof.

9. Peptide for use according to any one of claims 1 to 8, wherein the isolated peptide comprises at least one peptide having amino acid sequence (X_1"')_M"' - (X_2"')_O"' - X_3"' - (X_4"')_P"' - (X_5"')_Q"' -(X_6"')_T"' - (X_7"')_R"' - (X_8"')_S"' or the reverse sequence thereof, wherein
X_1"' is a hydrophobic amino acid, preferably selected from the group consisting of proline (Pro) and phenylalanine (Phe),
X_2"' is a basic amino acid, preferably arginine (Arg),
X_3"' is a hydrophobic amino acid, preferably tryptophan (Trp),
X_4"' is selected from the group consisting of alanine (Ala), arginine (Arg), glutamine (Gln), asparagine (Asn) and lysine (Lys),
X_5"' is selected from the group consisting of isoleucine (Ile), phenylalanine (Phe) and tryptophan (Trp),
X_6"' is selected from the group consisting of glutamine (Gln), arginine (Arg) and asparagine (Asn),
X_7"' is a hydrophobic amino acid, preferably selected from the group consisting of isoleucine (Ile), tryptophan (Trp) and phenylalanine (Phe), and
X_8"' is arginine (Arg), and wherein
M"' is 0, 1, 2 or 3,
T"' is 0, 1, 2 or 3,
O"' is 0 or 1,
P"' is 1, 2 or 3,
Q"' is 1 or 2, and
R"' and S"' are 0, 1 or 2.

10. Peptide for use according to claim 9, wherein the at least one peptide have an amino acid sequence selected from the group consisting of FWRNIRIRR (SEQ ID No. 72), FWQRIRIRR (SEQ ID No. 73), FWRWRIWR (SEQ ID No. 74), FWRIRIRR (SEQ ID No. 75), FWRNIRIWRR (SEQ ID No. 76) and FwRNIRIRR (SEQ ID No. 77) or the reverse sequence thereof.

11. Peptide for use according to any one of claims 1 to 10, wherein the isolated peptide comprises at least one peptide having an amino acid sequence selected from the group consisting of RFWQRNIRKVRR (SEQ ID No. 62), RFWQRNIRKYR (SEQ ID No. 63), PFWQRNIRKWR (SEQ ID No. 64), RFRWQRNIRKYRR (SEQ ID No. 65), RWKRINRQWF (SEQ ID No. 66), KRFCFKK (SEQ ID No. 67), KRFSFKKC (SEQ ID No. 68), KRWSWKK (SEQ ID No. 69), FRFSFKK (SEQ ID No. 70), RRFWFRR (SEQ ID No. 71), RFWQRNIRIRR (SEQ ID No. 78), RWQRNIRIRR (SEQ ID No. 79) and RRWFWRR (SEQ ID No. 86) or the reverse sequence thereof.

12. Peptide for use according to any one of claims 1 to 11, wherein the isolated peptide comprises at least one peptide having an amino acid sequence selected from the group consisting of FIWQRNIRKVR (SEQ ID No. 34), FIWRWRWR (SEQ ID No. 49) and RRIRINRQWF (SEQ ID No. 80) or the reverse sequence thereof.

13. Peptide for use according to any one of claims 1 to 12, wherein at least two peptides having an amino acid sequence as defined in any one of claims 4 to 13 or the reverse sequence thereof are fused directly or via a linker to each other.

14. Peptide for use according to any one of claims 1 to 12, wherein said isolated peptide comprises at least two, preferably two, peptides having the same amino acid sequence and being selected from the peptides having an amino acid sequence as defined in any one of claims 4 to 13 or the reverse sequence thereof, wherein the at least two peptides are fused directly or via a linker to each other.

15. Peptide for use according to any one of claims 1 to 12, wherein said isolated peptide comprises at least two, preferably two, peptides, wherein an at least one first peptide has an amino acid sequence as defined in any one of claims 4 to 13 and the at least one second peptide the reverse sequence thereof, wherein the at least two peptides are fused directly or via a linker to each other.

16. Peptide for use according to any one of claims 1 to 18, wherein the cancer is selected from solid and non-solid tumors, including metastases, preferably selected from the group consisting of melanoma, rhabdomyosarcoma, glioblastoma, colorectal cancer, breast cancer, lymphoma, prostate cancer, pancreatic cancer, renal cancer, ovarian cancer and lung cancer.

Drawing